CN1653079A

CN1653079A - Intercalator containing pseudonucleotide(s)

Info

Publication number: CN1653079A
Application number: CNA028282140A
Authority: CN
Inventors: 乌尔夫·贝克·克里斯滕森; 埃里克·比耶勒高·彼得森
Original assignee: Human Genetic Signatures Pty Ltd
Current assignee: Human Genetic Signatures Pty Ltd
Priority date: 2001-12-18
Filing date: 2002-12-18
Publication date: 2005-08-10
Anticipated expiration: 2022-12-18
Also published as: EP1468007A2; WO2003052134A3; WO2003052134A2; AU2002358462A1; WO2003052133A2; WO2003052133A3; WO2003052132A2; AU2002358464B2; WO2003052132A3; CN1653079B; AU2002361946A8; WO2003051901A2; AU2002358463A1; AU2002358464A1; WO2003051901A3; AU2002358463A8; AU2002358462A8; AU2002361946A1

Abstract

The present invention relates to intercalator pseudonucleotides. Intercalator pseudonucleotides according to the invention are capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue and they comprise an intercalator comprising a flat conjugated system capable of co-stacking with nucleobases of DNA. The invention also relates to oligonucleotides or oligonucleotide analogues comprising at least one intercalator pseudo nucleotide. The invention furthermore relates to methods of synthesising intercalator pseudo nucleotides and methods of synthesising oligonucleotides or oligonucleotide analogues comprising at least one intercalator pseudonucleotide. In addtition, the invention describes methods of separating sequence specific DNA(s) from a mixture comprising nucleic acids, methods of detecting a sequence specific DNA (target DNA) in a mixture comprising nucleic acids and/or nucleic acid analogues and methods of detecting a sequence specific RNA in a mixture comprising nucleic acids and/or nucleic acid analogues. In particular said methods may involve the use of oligonucleotides comprising intercalator pseudo nucleotides. The invention furthermore relates to pairs of oligonucleotides or oligonucleotide analogues capable of hybridising to one another, wherein said pairs comprise at least one intercalator pseudonucleotide. Methods for inhibiting a DNAse and/or a RNAse and methods of modulating transcription of one or more specific genes are also described.

Description

The pseudonucleus thuja acid that comprises intercalator

Invention field

The present invention relates to the field of synthesizing ribonucleotide quasi-molecule, described ucleotides molecule can embed in the main chain of nucleic acid or nucleic acid analog.Especially, the present invention relates to this synthesizing ribonucleotide quasi-molecule that comprises intercalator, be called intercalator pseudonucleus thuja acid here.

The invention still further relates to nucleic acid analog that comprises intercalator pseudonucleus thuja acid and the method for preparing the intercalator monomeric unit.

In addition, the present invention relates to separate or the method for target sequence specific DNA and the method that reduces nucleic acid analog oneself hybridization from nucleic acid mixture, improve the specific method of results of hybridization, and the method for adjusting melting temperature(Tm) difference between the different hybridisation events of the parallel analysis that randomly in same reactor, carries out.

Background of invention

Nucleic acid, such as DNA and RNA and a large amount of nucleic acid analogs, such as PNA, HNA, MNA, ANA, LNA, CAN, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, β-D-RNA and other can with their complementary strand specific hybrid.This specific recognition can be used for the existence of test example as the specific nucleic acid sequence that is used for diagnostic purpose.Some synthetic nucleic acid has the enhanced affinity to nucleic acid usually.Can promote check and analysis and the nucleic acid that has a high affinity for target nucleic acid in addition to can be used for many other purposes greatly for the high affinity of target nucleic acid, such as gene targeting and purification of nucleic acids.

Many regrettably present obtainable nucleic acids also have very high affinity for similar nucleic acid.Be very undesirable for many purposes.For example, some synthetic nucleic acid probe has the tendency of the weakening of forming and another complementary nucleic acid bonded hairpin loop.

In addition, most of nucleic acid and most of synthetic nucleic acid analog can not strictly be distinguished various nucleic acid, and promptly they are the same preferably in conjunction with complementary DNA and complementary RNA.

Though known for a period of time have relatively large difference in the three-dimensional structure of DNA/DNA duplex and DNA/RNA crossbred, and some enzymes, picture ribonuclease H (RNase H) can be discerned respectively, and generally speaking the oligonucleotide of chemically modified can not be distinguished ssRNA and ssDNA.

Usually, some nucleic acids such as HNA and LNA that contain modification in the sugar ring have the enhanced affinity for ssDNA and ssRNA.These modify preferably stabilising effect (HNA and LNA are respectively Δ TM+3 to+5 ℃ and+4 to+8 ℃) for ssRNA surpass stabilising effect to ssDNA (to HNA and LNA be respectively Δ Tm+1 arrive+3 ℃ with+3 to+5 ℃).Though it is reported, some modify be fully RNA optionally, the meaning refer to these oligonucleotide analogs will be only with RNA and not with DNA hybridization, these duplexs have lower melting temperature(Tm) than comparable non-modification crossbred.On the other hand, have only the report of minority about the oligonucleotide of DNA selective modification.

Have in the past twenties years of fluorescently-labeled nucleoside analog together with differentiating and the development of detection specificity nucleotide sequence novel method has attracted a lot of people's interest.Used many different fluorescent probes, and have been a kind of of the most normal use as the pyrenes that form many ring excitons fragrance bodies.Several acyclic nucleoside analogs that comprise pyrene have been described.

The synthetic ucleotides molecule that comprises intercalator has been described in the prior art:

US 5,446, and 578 have described the synthesizing ribonucleotide quasi-molecule that comprises fluorescence molecule, and described fluorescence molecule demonstrates with the different spectrum change of concentration, for example pyrene.Especially, document description with the fluorescence molecule deutero-nucleic acid on the phosphoric acid of this nucleic acid main chain or nucleic acid, described nucleic acid main chain or nucleic acid comprise the acyclic backbone monomers of being made up of 5 each atom between two phosphoric acid ester of the nucleic acid main chain that is connected to this fluorescence molecule.Document description the fluorescence molecule outside that should be arranged in the nucleic acid spiral make them can not embed the nuclear base of nucleic acid.In addition, being interpreted as hybridizing afterwards, the fluorescence of fluorescence molecule strengthens and must have cats product in order to reach this effect.Document both openly the double-helical stabilization of nucleic acid do not differentiate difference between RNA and the DNA yet.

People such as Yamana described in 1999 one comprise 2 in the central position '-oligonucleotide of O-(1-pyrenyl methyl) uridine.The oligonucleotide of described oligonucleotide and unmodified compares DNA to have higher affinity and RNA is had lower affinity.The fluorescence of monomer and exciplex strengthens after hybridization.

People such as Yamana have described the phosphoramidit that is connected with pyrene in 1997, described phosphoramidit can embed any desired position of nucleic acid.Especially described phosphoramidit can embed nucleic acid as the acyclic backbone monomers of being made up of 5 atoms between two phosphoric acid ester of nucleic acid main chain.After the hybridization, the nucleic acid that excimer fluorescence strengthens greatly and described phosphoramidite is embedded has kept DNA normally in conjunction with affinity.

People such as Korshun have described the phosphoramidit that is connected with pyrene in 1999, described phosphoramidit can embed any desired position of nucleic acid.Especially described phosphoramidit can embed nucleic acid as the acyclic main chain list matrix of being made up of 5 atoms between two phosphoric acid ester of nucleic acid main chain.In addition, document description the embedded oligonucleotide of described phosphoramidite and DNA is had higher affinity according to describing the oligonucleotide of oligonucleotide than unmodified.It is introduced because joint is shorter, so it is coplanar mutually near being suppressed greatly to be positioned at the sealing of two pyrene residues of oligonucleotide close position of modification.Excimer fluorescence strengthens after hybridization, yet comprises that when not hybridizing the oligonucleotide of 5 this pyrene pseudonucleus thuja acids finally also demonstrates stronger excimer fluorescence.

US 5,414, and 077 has described the pseudonucleus thuja acid that can comprise such as the intercalator of acridine or anthraquinone.The pseudonucleus thuja acid comprises organic main chain of an achiral or single enantiomer, such as diethanolamine.The pseudonucleus thuja acid can be in any desired position embeds oligonucleotide.Common this oligonucleotide has higher affinity for complementary nucleotide, especially when the pseudonucleus thuja acid embeds end.Document is not described fluorescence data.

US 6,031, and 091 has described the pseudonucleus thuja acid that can embed oligonucleotide in any position.Especially document description comprise and also mention the pseudonucleus thuja acid in the main chain of acyclic phosphorescent substance and the document and can comprise intercalator.The concrete pseudonucleus thuja acid of describing in the document comprises the very long joint that the poly aromatic body is connected to nucleic acid main chain.

EP 0 916 737 A2 have described with for example intercalation compound deutero-polynucleotide.Intercalation compound will be preferably between have about 10 Nucleotide.Polynucleotide can be derived at phosphoric acid, sugar or nuclear base portion.Especially, thus they can examine on the base not to be connected with the poly aromatic body derive by the long joint of 7 or 1 atoms with mode that Watson-Crick base pairing is conflicted.Describing in the document by embedding fluorescence intensity increases.

Strassler etc. have described in 1999 and have comprised pyrene for example but not the pseudonucleus thuja acid of the fluorescence molecule of nuclear base.

People such as Ebata have described 5 ' end of the Nucleotide intercalation of DNA oligonucleotide that pyrene is modified and the Nucleotide that pyrene is modified have been embedded another 3 ' end in nineteen ninety-five.By to hybridize to target sequence, produce excimer bands of a spectrum at the 490nm place from the tight each other approaching mode of the pyrene part of two chains.

People such as Paris have described disclosed system such as the people that is similar to Ebata in 1998, wherein said system can be used for detecting mispairing.Yet the ability that fully-complementary sequence (wt) and a single point sudden change (mut) are distinguished by system is hybridization and the ability of not hybridizing in another case under a kind of situation of a kind of probe due to.This just means that described phenomenon is temperature controlled and the length that limited probe has limited selectivity thus and control has proposed higher requirement to temperature.

Summary of the invention

The application U.S. Provisional Application No.60/365 that requirement was submitted on April 20th, 2002 according to § 119 (e), 545 right of priority, it all is incorporated herein by reference herein.

Whole patents and the non-references quoted in application or the application also all are incorporated herein by reference.

The present invention relates to the pseudonucleus thuja acid or the polynucleotide analogue that comprise intercalator and have one or more following features:

Can

1. embed double-helical predetermined position; And/or

2. strengthened affinity substantially to DNA; And/or

3. suppress or reduce self and cross hybridization; And/or

4. distinguish between the different nucleic acid, such as the difference between RNA and the DNA; And/or

5. strengthen the specificity of hybridization substantially; And/or

6. strengthen the stability of nuclease; And/or

7. strengthen the embedding of chain significantly; And/or

8. the hybridization back shows the change of fluorescence intensity.

Therefore there are a large amount of needs, that is to say that described pseudonucleus thuja acid can change the characteristic of oligonucleotide according to above-mentioned standard cheap pseudonucleus thuja acid.

One aspect of the present invention just provides common structure

X-Y-Q

Intercalator pseudonucleus thuja acid, wherein

X is the backbone monomers unit that can embed nucleic acid or nucleic acid analog main chain,

Q is the intercalator of the conjugate system that comprises that at least one is generally planar, and described conjugate system can be piled up altogether with the nucleoside base of DNA; And

Y is the shank that described backbone monomers unit is connected with described intercalator.

More preferably, the present invention relates to a common structure is

X-Y-Q

Intercalator pseudonucleus thuja acid, wherein

X be for can embed the nucleic acid of following general formula or the backbone monomers unit in the nucleic acid analog main chain,

N=1 to 6 wherein,

R is the phosphorus atom that trivalent or pentavalent replace,

R2 is independently selected from the atom that can form at least two keys, and R2 replaces separately, and

R6 is a protecting group.

Q is the intercalator of the conjugate system that comprises that at least one is generally planar, and described conjugate system can be piled up altogether with the nuclear base of DNA; And

Y is the shank of arbitrary R2 of connection described backbone monomers unit and described intercalator; And

Wherein the total length of Q and Y at 7  in the scope of 20 ,

Precondition is when intercalator is pyrene, the total length of Q and Y at 9 in the scope of 13 , preferably from 9 to 11 .

Term " embeds the main chain of nucleic acid or nucleic acid analog " and is meant that intercalator pseudonucleus thuja acid can embed in the sequence of nucleic acid and/or nucleic acid analog.

Term " planar conjugate system " is meant that all whole atoms that are included in the conjugate system are positioned on the plane.Term " generally planar conjugate system " is meant that 20% all atoms that are included in the conjugate system are not to be positioned at any time on the described plane at the most.

Term " conjugate system " is meant (technical term of chemistry summary in 1987 such as Gold (the Compendium of Chemical Terminology of structure unit of the chemical bond that comprises the atom p Orbital Overlap with three or more adjacent atoms, Blackwell ScientificPublications, Oxford, UK).

The of the present invention accumulation altogether is the abbreviation of coaxial accumulation.Coaxial accumulation is a structure favourable on the energy, and wherein planar molecule is similar to packed structures along coaxial arrange (flat edge opposite planar limit) on surface each other.Interaction of piling up altogether between two πDian Zi clouds that need independent molecule of the present invention.In duplex under intercalator pseudonucleus thuja acid and the nuclear base situation of piling up altogether, preferably on opposite strand with the πDian Zi system interaction, more preferably on two chains with the πDian Zi system interaction.All found common accumulation at intermolecular and intramolecularly.For example nucleic acid adopts double-spiral structure that the nuclear base is piled up altogether.

A second aspect of the present invention provides synthetic this intercalator pseudonucleus thuja acid ground method, wherein synthetic can comprising the steps:

A1) provide the compound that comprises intercalator, described intercalator comprises at least aly can pile up the shank that generally planar conjugate system and optional is connected to active group altogether with the nuclear base of nucleic acid; With

B1) provide a tab precursor molecule that comprises at least two active groups, described two active groups can randomly be protected respectively; With

C1) thus the reaction of described intercalator and described tab precursor is obtained one intercalator-joint; With

D1) provide a backbone monomers precursor unit that comprises at least two active groups, described two active groups can randomly be protected respectively and/or shelter) and randomly comprise a shank; With

E1) with described intercalator-joint and described backbone monomers precursors reaction and obtain one intercalator-joint-backbone monomers precursor; Perhaps

A2) provide a backbone monomers precursor unit that comprises at least two active groups, described two active groups can randomly be protected respectively and/or shelter) and randomly comprise a shank; And

B2) provide a tab precursor molecule that comprises at least two active groups, described two active groups can randomly be protected respectively; With

C2) thus described monomer precursor unit is obtained one main chain-joint with the reaction of described tab precursor; And

D2) provide a compound that comprises intercalator, described intercalator comprises at least one generally planar conjugate system and a shank that is connected to active group randomly, and described planar conjugate system can pile up altogether with the nuclear base of nucleic acid; With

E2) with described intercalator and described main chain-joint reaction and obtain one intercalator-joint-backbone monomers precursor; Perhaps

A3) provide a compound that comprises intercalator, described intercalator comprises that conjugate system and that at least one is generally planar is connected to the shank of active group, described conjugate system can with the nuclear base and the common accumulation of nucleic acid; With

B3) provide the backbone monomers precursor unit that comprises at least two active groups and a shank, described two active groups can randomly be protected respectively and/or shelter); And

C3) with the reaction of described intercalator shank and described backbone monomers precursor joint and obtain one intercalator-joint-backbone monomers precursor;

And

F) randomly protect and/or go to protect described intercalator-joint-backbone monomers precursor; And

G) provide a phosphorescent substance its comprise the compound that two pseudonucleus thuja acids, Nucleotide and/or nucleotide analogs can be coupled together; And

H) with described P contained compound and described intercalator-joint-backbone monomers precursors reaction; And

I) obtain an intercalator pseudonucleus thuja acid

A third aspect of the present invention provides oligonucleotide or the oligonucleotide analogs that comprises at least a intercalator pseudonucleus thuja acid of the present invention, comprises at least a formula such as one

The oligonucleotide or the oligonucleotide analogs of the intercalator pseudonucleus thuja acid of X-Y-Q, wherein

X is the main chain that a backbone monomers unit can embed nucleic acid or nucleic acid analog,

Q one comprises the intercalator of at least a generally planar conjugate system, and described conjugate system can be piled up altogether with the nuclear base of DNA; And

Y is the shank of a connection described backbone monomers unit and described intercalator.

Another aspect of the present invention provides to synthesize and comprises the oligonucleotide of at least a intercalator pseudonucleus thuja acid or the method for oligonucleotide analogs, and wherein said method comprises the steps:

A) intercalator pseudonucleus thuja acid of the present invention is contacted with the upholder bonded Nucleotide, oligonucleotide, nucleotide analog and/or the oligonucleotide analogs chain that are increasing; And

B) with described intercalator pseudonucleus thuja acid and described upholder bonded Nucleotide, oligonucleotide, nucleotide analog or oligonucleotide analogs reaction; And

C) randomly by increasing one or more Nucleotide, nucleotide analog or intercalator pseudonucleus thuja acid oligonucleotide and/or further described oligonucleotide and/or the oligonucleotide analogs of prolonging of oligonucleotide analogs to the expectation sequence; And

D) described oligonucleotide and/or oligonucleotide analogs are separated from described solid support; And

E) obtain to comprise the described oligonucleotide and/or the oligonucleotide analogs of at least a intercalator pseudonucleus thuja acid thus.

Another aspect of the present invention provides oligonucleotide or the oligonucleotide analogs that comprises at least a intercalator pseudonucleus thuja acid of the present invention, and wherein the melting temperature(Tm) of the duplex of being made up of described oligonucleotide or oligonucleotide analogs and complementary DNA (DNA crossbred) is obviously higher than the melting temperature(Tm) of the crossbred of being made up of the described oligonucleotide analogs that comprises an at least a intercalator and a complementary RNA (RNA crossbred).

Equally, embed at least a intercalator pseudonucleus thuja acid of the present invention cause in oligonucleotide or the oligonucleotide analogs the described oligonucleotide that has a pseudonucleus thuja acid and a complementary oligonucleotide or oligonucleotide analogs duplex melting temperature(Tm) with.

Equally, an aspect of of the present present invention provides oligonucleotide and/or the oligonucleotide analogs that comprises at least a intercalator pseudonucleus thuja acid, wherein the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide and/or oligonucleotide analogs and complementary DNA (DNA crossbred) is than obviously higher by the melting temperature(Tm) of the molectron of the oligonucleotide that lacks intercalator pseudonucleus thuja acid and/or oligonucleotide analogs and described complementary DNA, and described oligonucleotide and/or oligonucleotide analogs are made up of the nucleotide sequence identical with described oligonucleotide and/or oligonucleotide analogs.

In addition, an aspect of of the present present invention provides oligonucleotide and/or the oligonucleotide analogs that has comprised at least a intercalator pseudonucleus thuja acid, wherein the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide analogs and complementary RNA (RNA crossbred sequence) is than obviously higher by the melting temperature(Tm) of the crossbred of the oligonucleotide of described shortage intercalator pseudonucleus thuja acid and/or oligonucleotide analogs and described complementary RNA, and described oligonucleotide and/or oligonucleotide analogs are made up of the nucleotide sequence identical with described oligonucleotide analogs.

Intercalator pseudonucleus thuja acid can be arranged in any suitable position of oligonucleotide, and according to the RNA sequence, intercalator pseudonucleus thuja acid is preferably placed at an end or the two ends of oligonucleotide analogs.

Another aspect of the present invention has provided from the method for the mixture separation sequence specific DNA that comprises nucleic acid, and described method comprises the steps:

A) provide a mixture that comprises nucleic acid; And

B) provide one or more to comprise the different oligonucleotide or the oligonucleotide analogs of at least a intercalator pseudonucleus thuja acid, wherein the melting temperature(Tm) of the crossbred of being made up of the described oligonucleotide that comprises at least a intercalator pseudonucleus thuja acid or oligonucleotide analogs and a homology complementary dna sequence (DNA crossbred) is obviously higher than the melting temperature(Tm) of the crossbred of being made up of the described oligonucleotide that comprises at least a intercalator pseudonucleus thuja acid or oligonucleotide analogs and a homology complementary RNA (RNA crossbred), and can hybridize with described sequence specific DNA comprising the described oligonucleotide or the oligonucleotide analogs of at least a intercalator pseudonucleus thuja acid; And

C) described mixture and described oligonucleotide or oligonucleotide analogs are incubated under the condition of described oligonucleotide or the similar and described sequence specific DNA of oligonucleotide (DNA crossbred) hybridization; And

D) from mixture separation oligonucleotide or oligonucleotide analogs together with the nucleic acid that hybridizes to described oligonucleotide; And

Obtain the mixture that isolating sequence specific DNA and isolating residue comprise nucleic acid thus.

Another aspect of the present invention has provided the method that detects sequence-specific RNA in the mixture that comprises nucleic acid and/or nucleic acid analog, and described method comprises the steps:

A) provide a nucleic acid mixture; And

B) provide one or more to comprise the different oligonucleotide or the oligonucleotide analogs of at least a intercalator pseudonucleus thuja acid, wherein the melting temperature(Tm) of the crossbred of being made up of the described oligonucleotide that comprises at least a intercalator pseudonucleus thuja acid or oligonucleotide analogs and a homology complementary dna sequence (DNA crossbred) is obviously higher than the melting temperature(Tm) of the crossbred of being made up of the described oligonucleotide that comprises at least a intercalator pseudonucleus thuja acid or oligonucleotide analogs and a homology complementary RNA (RNA crossbred), and comprising described oligonucleotide or the oligonucleotide analogs and the complementation basically of described sequence-specific RNA of at least a intercalator pseudonucleus thuja acid; And

C) provide one comprise detectable label and one can with the probe of the nucleotide sequence of described sequence-specific RNA hybridization; And

D) described mixture and described oligonucleotide or oligonucleotide analogs are incubated under the condition of hybridization; And

E) described mixture and described probe are incubated under hybridization conditions; And

F) detect described detectable label; And

Detect described sequence-specific RNA thus.

An aspect of of the present present invention provides oligonucleotide or the oligonucleotide analogs that comprises at least a intercalator pseudonucleus thuja acid of the present invention in addition, and wherein the melting temperature(Tm) of self crossbred of being made up of described oligonucleotide or oligonucleotide analogs is obviously lower than the melting temperature(Tm) of the heterozygote crossbred of being made up of the described described oligonucleotide that comprises at least a intercalator or oligonucleotide analogs and a homology complementary DNA (DNA crossbred).

In addition, an aspect of of the present present invention provides and improves the specific method of hybridization between the oligonucleotide comprise at least a intercalator pseudonucleus thuja acid or oligonucleotide analogs and a complementary nucleic acid target or the nucleic acid analog target, and the crossbred of wherein said oligonucleotide or oligonucleotide analogs and described target has than described oligonucleotide analogs and the nucleic acid different with described target and/or the obvious higher melting temperature(Tm) of crossbred of nucleic acid analog.

Equally, another aspect of the present invention provides multiple crossing and adjusts different IPs thuja acid and/or oligonucleotide analogs sequence and the complementary and method of melting temperature(Tm) between its homology complementary nucleic acid and/or the nucleic acid analog target randomly thereof in analyzing, at least two oligonucleotide or oligonucleotide analogs comprise at least one intercalator pseudonucleus thuja acid in wherein said different IPs thuja acid and/or the oligonucleotide analogs sequence, between wherein said oligonucleotide and/or oligonucleotide analogs and the described target melting temperature(Tm) of crossbred than same sequence do not have the described oligonucleotide of intercalator pseudonucleus thuja acid and/or oligonucleotide analogs and described target the obvious homology more of melting temperature(Tm).

In addition, an aspect of of the present present invention provides and comprises that at least one embeds the oligonucleotide or the oligonucleotide analogs of pseudonucleus thuja acid, and it does not comprise that than corresponding the corresponding oligonucleotide of intercalator pseudonucleus thuja acid is more stable significantly to nuclease.

Another aspect of the present invention provides and comprises that at least a fluorescence embeds the oligonucleotide and/or the oligonucleotide analogs of pseudonucleus thuja acid, wherein said oligonucleotide or oligonucleotide analogs can be hybridized with complementary DNA, and wherein said hybridization causes the reduction of described oligonucleotide and/or oligonucleotide analogs fluorescence intensity.Therefore, fluorescent characteristic can be used for detecting the described oligonucleotide that comprises at least a intercalator pseudonucleus thuja acid and/or the hybridization between oligonucleotide analogs and the described complementary DNA.

In addition, one aspect of the present invention just provides a pair of oligonucleotide or the oligonucleotide analogs that comprises first sequence, described first sequence is oligonucleotide and/or the oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid, and second sequence can with described first sequence hybridization, comprising the definition of the oligonucleotide of first sequence or oligonucleotide analogs as above.

Equally, one aspect of the present invention just provides the method that suppresses deoxyribonuclease and/or rnase, thereby described at least a oligonucleotide and/or the oligonucleotide analogs that will contain at least one intercalator pseudonucleus thuja acid of comprising is delivered to the activity that suppresses described deoxyribonuclease and/or rnase in rnase and/or the deoxyribonuclease.

Relate to the method that one or more specific genes of adjusting are transcribed on the other hand in invention, described method comprises the steps:

A) provide a re-reading system;

B) provide at least a aforesaid oligonucleotide analogs,

And wherein said oligonucleotide and/or oligonucleotide analogs can be regulated the complementary strand hybridization that sequence or described gene and/or its are regulated sequence with described gene and/or its; And

C) described oligonucleotide and/or oligonucleotide analogs are introduced re-reading system; And

D) allow oligonucleotide and/or oligonucleotide analogs and described one or more genes and/or its to regulate sequence or gene and/or its and regulate the hybridization of the complementary strand of sequence; And

Thereby regulate described gene transcription.

Final an aspect of of the present present invention is that the method with two kinds or more of preferably great majority or all characteristics and foregoing description is combined in method and the product that obtains to have favourable function and hybridization correlated characteristic in the novel method.

Description of drawings

Fig. 1 illustrates a kind of intercalator pseudonucleus thuja acid, i.e. synthesizing with 5 phosphoramidites of representing.

Fig. 2 illustrate in " MacroModel ", have sequence 5 '-the double-helical Structure Calculation of self complementary DNA of XCGCGCG-3 ', X=pyrene unit.Pyrene part and the coaxial accumulation of following base pair.

Fig. 3 illustrate duplex 5 '-AGCTTGCCTTGAG-3 '+5 '-predict of CTCAAGXCAACCT-3 ', X=5.The coaxial accumulation of contiguous nuclear base up and down of the relative chain of pyrene.

Fig. 4 illustrate have sequence 5 '-AGCTTGCTTGAG-3 '+5 '-CTCAAGXCAACCT-3 ', the double-helical predict of 12/13-aggressiveness of X=5 (Fig. 1), X=5.The pyrene part can be done mutually with the contiguous up and down nuclear base of opposite strand.Distance indication between nuclear base and the pyrene part is on the right side.

Fig. 5 illustrates the fluorescence measurement of the poly-ssDNA (★) of 13-that embeds single pyrene; It is with complementary, and 12-gather the duplex that the duplex of RNA () and it and complementary 12-gather DNA (◆).Sequence is identical with the sequence that table 3 shows.

Fig. 6 illustrates the fluorescence measurement of the poly-ssDNA of 14-with two pyrene inserts that separated by a Nucleotide (★); The duplex of the poly-RNA () of itself and complementary 12-and it and complementary 12-gather the duplex of DNA (◆).

Fig. 7 illustrates a method for preparing the RT-PCR sample.

Fig. 8 illustrates a method for preparing the RT-PCR sample.

Fig. 9 illustrates a method for preparing the RT-PCR sample.

Figure 10 illustrates the method for preparation one sequence specific DNA.

Figure 11 illustrates the method for preparation one sequence specific DNA.

Figure 12 illustrates the method for utilizing the chip detection sequence specific DNA.

Figure 13 illustrates the various oligonucleotide that can be used as probe on the chip.

Figure 14 illustrates the quantitative analysis of PCR.

Figure 15 illustrates the blocking-up of transcribing that a pair of oligonucleotide that utilizes the present invention to be shown as A and B respectively carries out.

Figure 16: the nuclease resistance of two kinds of oligonucleotide, one of them comprises the double-helical nuclease resistance of intercalator pseudonucleus thuja acid (INA oligonucleotide) and described two oligonucleotide.

Figure 17: the secondary structure that forms the hair clip of probe 1.Monomer and excimer fluorescence are quenchers in this structure.

Figure 18: the secondary structure of probe I when hybridizing to target sequence.When hybridizing to target sequence, any formation of excimer mixture can be observed excimer fluorescence thus.Monomeric fluorescence intensity strengthens equally.

Figure 19 is observable SYBR green 11 painted INA oligonucleotide on the ArrayWorx scanner.

Figure 20: illustrate the test that is combined in the oligonucleotide on the Asper SAL slide glass.

Figure 21: the exciplex fluorescence when being positioned between molecule X and the Y near nearest neighbour position (sequence I).

Figure 22: the exciplex fluorescence when being positioned at close position (sequence II) between molecule X and the Y.

Figure 23: the exciplex fluorescence when at close position between molecule Y and the Z.

Figure 24: illustrate EtBr dyeing.

Figure 25: the sequence of the double-stranded target oligonucleotide of use, attack lOs and complementary pairing IOs.Y represents to embed the unit.

Figure 26: lOs is spontaneous in conjunction with target DNA.

Being reflected at 37 ℃ carried out 1 hour in the 20 μ l that containing 126nM IO under the condition that contains or do not contain the 20nM target DNA.Observe by the combination of electrophoretic analysis on 10% polyacrylamide/1/2 * TBE gel and by the phosphorescence imaging.

Figure 27: the IO-DNA mixture forms needs the sequence complementation.

Under 37 ℃ of conditions that contain or do not contain 20nM target DNA (strand or two strands), reaction is 2 hours under the volume of the 15 μ l of the IO that contains display density.Observe by the combination of electrophoretic analysis on 10% polyacrylamide/1/2 * TBE gel and by the phosphorescence imaging.

Figure 28: the IO pairing in the spontaneous target combination.

Reaction is 2 hours under the volume of the 15 μ l of the IO of 37 ℃ of preannealing P32-marks that contain 20nM target DNA and display density.Observe by the combination of electrophoretic analysis on 10% polyacrylamide/1/2 * TBE gel and by the phosphorescence imaging.

Figure 29: pairing does not influence spontaneous bonded efficient.Reaction is 4 hours under the volume of 37 ℃ of 15 μ l that contain 20nM target DNA and shown increasing amount IOs (40-80-160nM).Observe by the combination of electrophoretic analysis on 10% polyacrylamide/1/2 * TBE gel and by the phosphorescence imaging.Band intensity is the relative value of expression bands of a spectrum district intensity.

Figure 30: the formation of IO-DNA mixture in nuclear extract

Under the volume of 15 μ l of 180nM IO that contains preannealing and shown 20nM target DNA, react and in reactant, add nuclear extract (NE) as shown in the figure.Reactant is 37 ℃ of insulations 10 minutes, and then adds warm 60 minutes of the follow-up continuation of insurance of Proteinase K of the 10%SDS of 1.125 μ l and 37.5 μ g.Observe by the combination of electrophoretic analysis on 7% polyacrylamide/1/2 * TBE gel and by the phosphorescence imaging.

Figure 31: nf forms promoting the IO-DNA mixture by IO

Under the volume of the 15 μ l that contain 180nM IO and 20nM target DNA, react.The HeLa nuclear extract that in reactant, adds 10 μ g.Reactant is 37 ℃ of insulations 10 minutes, and then adds warm 60 minutes of the follow-up continuation of insurance of Proteinase K of the 10%SDS of 1.125 μ l and 37.5 μ g.Observe by the combination of electrophoretic analysis on 10% polyacrylamide/1/2 * TBE gel and by the phosphorescence imaging.

The chemical structure of Figure 32: LNA and INA P nucleotide monomer.B=examines base.

Figure 33: when with complementary structure and the hybridization of LNA target, have the melting temperature(Tm) data of the INAs of various embedding collection of illustrative plates.P=INA monomer P.T ^LWith ^MeC ^LBe respectively thymus pyrimidine and 5-methylcytosine fixed Nucleotide.

Figure 34: transition temperature, the Tm of hair clip probe and ssDNA target (℃).T ^LWith ^MeC ^LBe respectively thymus pyrimidine and 5-methylcytosine fixed Nucleotide.

Figure 35: A) comprise the transition curve B of the non-embedding pseudonucleus thuja acid of probe) two kinds comprise that one embeds the reference duplex of the LNA probe of pseudonucleus thuja acid together with unmodified.C) comprise the reference duplex of the LNA probe of one or two embedding pseudonucleus thuja acid together with unmodified.D) comprise that a non-embedding pseudonucleus thuja acid of LNA probe and two comprise that one embeds the probes of pseudonucleus thuja acid, together with corresponding all hybridize to and comprise that one embeds the dna probe of the target sequence of pseudonucleus thuja acid.

Figure 36: schema 1.At transition temperature by T ₄The diagram of the structure that-LNA oligonucleotide forms.

Figure 37: 1 '-azepine pyrene methyl pseudonucleus thuja acid synthetic.

Figure 38: sequence and the hybridization data of synthetic ODNs in DNA/DNA (RNA) duplex.

The hybridization data that Figure 39: DNA three approach engage.

Figure 40: illustrate a sign primer.

Figure 41: illustrate the PCR quantitative analysis strategy that utilizes the sign primer.

Figure 42: illustrate complete complementarity and mispairing/excimer and form.

Detailed Description Of The Invention

Nucleic acid

Term " nucleic acid " comprises naturally occurring nucleic acid, DNA and RNA, comprises the derivative of naturally occurring DNA and RNA, such as, but not limited to methylate DNA, comprise the DNA of adducts and be covalently bound to proteic RNA.Term " nucleic acid analog " comprises natural synthesis of derivatives and the analogue that has nucleic acid, DNA and RNA.The synthetic analogue comprises one or more nucleotide analogs.Term " nucleotide analog " comprises that all can be incorporated into nucleic acid main chain and nucleotide analog that can specificity base pairing (vide infra), basically as naturally occurring cyclic nucleotide.

Therefore the arbitrary molecule of forming by many Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid basically of term " nucleic acid " or " nucleic acid analog " expression.Intercalator pseudonucleus thuja acid is described in detail hereinafter.Nucleic acid of the present invention or nucleic acid analog can comprise more kinds of different IPs thuja acid and the nucleotide analog with various backbone monomers unit (vide infra).

Preferably, the strand of nucleic acid of the present invention or nucleic acid analog can be with complementary single-chain nucleic acid and/or nucleic acid analog be hybridized formation double-strandednucleic acid or nucleic acid analog substantially.More preferably, so a kind of double-stranded analogue can form a duplex.Preferably, because hydrogen bonding forms duplex, more preferably duplex is the duplex that is selected from A form, B form, Z-shaped formula and intermediate thereof.

Therefore, institute's nucleic acid of the present invention and nucleic acid analog include but not limited to the nucleic acid and/or the nucleic acid analog of following kind: DNA, RNA, PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, β-D-RNA and composition thereof and its crossbred, with and phosphorus atom modify, such as, but not limited to thiophosphatephosphorothioate, methyl phospholates, phosphoramidiates, phosphorodithiates, the seleno phosphoric acid ester, phosphotriester and phosphoboranoates.Do not wrap the phosphorated compound in addition and can be used for connecting Nucleotide, described P contained compound is such as, but not limited to methyl-imino methyl, formacetate, thioformacetate and the linking group that comprises acid amides.Especially nucleic acid and nucleic acid analog can comprise one or more intercalator pseudonucleus thuja acids of the present invention.

Be meant at context " mixture " and comprise nucleic acid or the nucleic acid analog chain that contains various Nucleotide or nucleotide analog.In addition, be meant at context " crossbred " and comprise that a chain contains Nucleotide with one or more main chains or nucleotide analog and another chain comprises the Nucleotide with various main chains or the nucleic acid or the nucleic acid analog of nucleotide analog.Term " duplex " is meant the hybridization product of two nucleic acid and/or nucleic acid analog, and its medium chain is preferably the nucleic acid and/or the nucleic acid analog of identical type.

HNA is meant the nucleic acid of being described nineteen ninety-five by people such as for example Van Aetschot.MNA is meant the nucleic acid of being described in 1998 by people such as Hossain.ANA is meant the nucleic acid of being described in 1999 by people such as Allert.LNA can be arbitrary LNA molecule of WO 99/14226 (Exiqon) description, and preferably, LNA is selected from the molecule of describing in the summary of WO 99/14226.More preferably, LNA is Singh etc. 1998, the nucleic acid that Koshkin etc. 1998 or Obika etc. described in 1997.PNA is meant the peptide nucleic acid(PNA) by descriptions in 1991 such as for example Nielsen.

Term Nucleotide is represented the assembled unit of nucleic acid or nucleic acid analog, and term Nucleotide comprises naturally occurring Nucleotide and derivative thereof and can fulfil Nucleotide with naturally occurring Nucleotide and derivative identical function thereof basically.Naturally occurring ringed nucleus thuja acid comprises that containing four kinds examines bases adenine (A), thymus pyrimidine (T), the deoxyribonucleotide of one of guanine (G) or cytosine(Cyt) (C) and contain four kinds nuclear bases adenine (A), uridylic (U), the ribonucleotide of one of guanine (G) or cytosine(Cyt) (C).

Nucleotide analog may be to embed nucleic acid main chain and arbitrary ucleotides molecule that can the specificity base pairing.

Non-natural of the present invention exists ground Nucleotide to include but not limited to be selected from following Nucleotide PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, β-D-RNA.

The functional group of Nucleotide of the present invention and nucleotide analog can do mutually by the nuclear base ground hydrogen bond specificity of described mutual not Nucleotide with complementary nucleotide and can embed nucleic acid or nucleic acid analog.Naturally occurring Nucleotide and some nucleotide analogs can embed nucleic acid or nucleic acid analog by enzymatic, and for example by RNA or archaeal dna polymerase, yet Nucleotide or nucleotide analog also may embed nucleic acid or nucleic acid analog by chemistry.

Nucleic acid or nucleic acid analog can be prepared by two are coupled on another than small nucleic acids or nucleic acid analog in addition, for example the enzymatic reaction that this process can linked enzyme or finish by chemical reaction.

Nucleotide or nucleotide analog comprise a backbone monomers unit and a nuclear base.The nuclear base can be naturally occurring nuclear base or its derivative or its derivative or its analogue that can fulfil identical function basically.The function of nuclear base is to be connected by the hydrogen bond specificity with one or more other nuclear base.Therefore a kind of key character of examining base is exactly that it can only form stable hydrogen bond with one or several other nuclear base, but can not form stable hydrogen bond with most other nuclear base that comprises himself usually.A kind of specificity interaction so-called " base pairing " of examining base and another kind of nuclear base.

Base pairing produces the specific hybrid between predetermined and the complementary nucleotide.Complementary nucleotide of the present invention is the Nucleotide that comprises nuclear base that can base pairing.

In the naturally occurring nuclear base, VITAMIN B4 (A) and thymus pyrimidine (T) or uridylic (U) pairing; Guanine (G) and cytosine(Cyt) (C) pairing.Therefore, for example comprise that the Nucleotide of A is complementary to the Nucleotide that comprises T or U, comprise that the Nucleotide of G is complementary to the Nucleotide that comprises C.

Nucleotide of the present invention can further be derived and be comprised an additional molecular entity.Nucleotide can be derived on the nuclear base or on the backbone monomers unit.The site of preferably deriving on the base comprises the VITAMIN B4 in 8-site, the uridylic in 5-site, the cytosine(Cyt) in 5-or 6-site and the guanine in 7-site.Especially, the methylation in cytosine(Cyt) 5 sites is important, and therefore a preferred embodiment of the present invention is to distinguish some or all of methylated sequence and the non-sequence that methylates.Heterocycle is modified can be grouped into three structural classes: enhanced base stacking, additional hydrogen bonding and their combination.The modification that π-electronic cloud by the expansion planar system increases base stacking is modified expression by the conjugation lipotropy in the 7-site of pyrimidine 5-site and 7-deaza purine.Modification substituting group in pyrimidine 5-site comprises propine, hexin, thiazole and simple methyl; The substituting group in the 7-site of 7-deaza purine comprises iodo, proyl and cyano group.Also can be and modify the 5-site of cytosine(Cyt)s to three ring condensed ring systems from propine to the 5-heterocycle, described three ring condensed ring systems derive from 4-and 5-site (cytosine(Cyt) folder).Second type heterocycle is modified and is represented that by 2-amino-VITAMIN B4 wherein additional amino provides another hydrogen bond in the A-T base pair, is similar to three hydrogen bonds in the G-C base pair.Provide the heterocycle of combined effect to modify by the andenine of 2-amino-7-deaza-7-modification and three ring cytosine(Cyt) analogues with oxyethyl group amido functional group of a heteroduplex.In addition, the oligonucleotide modified of the 2-aminoadenine modified of N2-is a kind of in the common modification.It is disconnected carbon location C-2 ' and the modification of C-4 ' that ribose or ribodesose are partly gone up the deutero-preferred sites, connect carbon C-1 ', the modification of C-3 ' and C-5 ', the displacement of glycosyloxyization, O-4 ', anhydrosugar is modified (the structure restriction), cyclohexanol is modified (the structure restriction), the change of ribofuranose ring size, connection site-sugar to sugar, (C-3 ' to C-5 '/C-2 ' to C-5 '), the sugar that heteroatomic ring is modified and the combination of above-mentioned modification.Yet also can derive in other site, as long as whole base pairing specificitys of nucleic acid or nucleic acid analog are not destroyed.At last, when the backbone monomers unit comprises a phosohate group, can derive the unitary phosphoric acid ester of some backbone monomers.

Here oligonucleotide of Shi Yonging or oligonucleotide analogs are the molecule that consists essentially of the sequence of Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.Preferably oligonucleotide or oligonucleotide analogs comprise 3-200,5-100,10-50 independent as defined above Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.

Target nucleic acid

Target nucleic acid or target nucleic acid analogue sequence be meant and comprise that one or more hybridizes one or more oligonucleotide and/or oligonucleotide analogs, for example the Nucleotide of the site/sequence of primer or probe or nucleotide analog sequence.Target sequence can be in arbitrary nucleic acid or nucleic acid analog including but not limited to other probe, RNA, genomic dna, plasmid DNA, cDNA, and can for example comprise that a wild-type or mutator gene sequence or its are regulated sequence or the amplifying nucleic acid sequence during for example by pcr amplification.Target sequence can be arbitrary length.Selected site may be or may not be a contiguous sequence.For example described site can be by two or more by forming in abutting connection with subsequence that many Nucleotide and/or nucleotide analog are separated.Preferably, the total length in the selected site of being made up of all subsequences that pass through described oligonucleotide and/or oligonucleotide analogs on particular target nucleic acid or the target nucleic acid analogue is generally less than 100 Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.

Homologous nucleic acid

Nucleic acid, homology complementation when nucleic acid analog, oligonucleotide or oligonucleotide analogs can be hybridized when them.Preferably homology complementary nucleic acid, nucleic acid analog, oligonucleotide or oligonucleotide analogs can hybridize under low stringency condition, more preferably homology complementary nucleic acid, nucleic acid analog, oligonucleotide or oligonucleotide analogs can be hybridized under medium stringent condition, and more preferably homology complementary nucleic acid, nucleic acid analog, oligonucleotide or oligonucleotide analogs can be hybridized under the height stringent condition.

Here the height stringent condition of Shi Yonging should represent with usually with for example by SouthernE.M., 1975, J.Mol.Biol, the Southern trace that 98:503-517 describes is compared strictness or the same with above-mentioned condition at least strict with hybridization about the condition that adopts.For this purpose, implement to comprise the step of prehybridization and hybridization usually.This step is utilized usually and is comprised 6 * SSPE, 5%Denhardt ' s, 0.5%SDS, 50% methane amide, the solution of 100 μ g/ml sex change salmon sperm dnas (cultivating 18 hours) at 42 ℃, succeeded by cleaning (at room temperature and at 37 ℃) with 2 * SSC and 0.5%SDS and with 0.1 * SSC and 0.5%SDS (cultivating 30 minutes) cleaning at 68 ℃, be described in " molecular cloning lab guide (Molecular Cloning/A Laboratory Manual) " cold spring port as Sambrook etc. 1989), it is incorporated herein by reference herein.

Here the medium stringent condition of Shi Yonging should be illustrated in and comprise 1mM EDTA, 10mMNa ₂HPO ₄H ₂O, 140mM NaCl are in the damping fluid of pH7.0 or be similar to and thisly have in the damping fluid of approximately identical influence and hybridize hybridizing strictness.Every kind of nucleic acid or the nucleic acid analog chain of about 1,5 μ M preferably, are provided.Perhaps, medium strictness can be illustrated in the KCl that contains 50mM, 10mM TRIS-HCl (pH9,0), and 0.1%Triton X-100 is hybridized in the damping fluid of 2mM MgCl2.

Low stringency condition of the present invention is illustrated in and contains 1M NaCl, 10mM Na ₃PO4 is in the damping fluid of pH7.0 or be similar to and thisly have in the damping fluid of about same affect and hybridize hybridizing strictness.

Perhaps, homology complementary nucleic acid, nucleic acid analog, oligonucleotide or oligonucleotide analogs are complimentary to one another substantially with a given sequence, such as surpassing 70% complementation, for example surpass 75% complementation, such as surpassing 80% complementation, for example surpass 85% complementation, such as surpassing 90% complementation, for example surpass 92% complementation, such as surpassing 94% complementation, for example surpass 95% complementation,, for example surpass 97% complementary nucleic acid, nucleic acid analog, oligonucleotide or oligonucleotide analogs such as surpassing 96% complementation.

Preferably described given sequence is long at least 4 Nucleotide, at least 10 Nucleotide for example, such as at least 15 Nucleotide, at least 20 Nucleotide for example, such as at least 25 Nucleotide, at least 30 Nucleotide for example are such as between 10 and 500 Nucleotide, for example between 4 and 100 Nucleotide are long, such as between 10 and 50 Nucleotide are long.More preferably, homology complementary oligonucleotide or oligonucleotide analogs and their whole length homology complementation basically.

The hybridization specificity

The hybridization specificity of nucleic acid and/or nucleic acid analog and/or oligonucleotide and/or oligonucleotide analogs is meant under given stringent condition according to the ability of the complementary hybridization of the described results of hybridization difference of their sequence difference homology to forming.Even usually purpose is that they and described target sequence have stronger similarity, a particular sequence (target sequence) and avoid hybridization with other sequence only in the mixture of target nucleic acid and/or nucleic acid analog and/or oligonucleotide and/or oligonucleotide analogs only.Sometimes the difference of among the target of the sequence area that is used for hybridizing and non-target sequence, having only a kind of or a few Nucleotide.

Here the hybridization high degree of specificity of Shi Yonging is illustrated in the hybridization down of height stringent condition, oligonucleotide or oligonucleotide analogs will be hybridized with the homologous target sequence under the described conditions, be better than significantly with described target sequence to have only one or the different sequence much at one of a few base replacement.

Difference

Difference be meant oligonucleotide and/or oligonucleotide analogs with sequence independently mode preferably with the ability of RNA or DNA hybridization.Therefore, comprise that the crossbred of oligonucleotide and/or oligonucleotide analogs and a homology complementary RNA (RNA crossbred) is higher significantly or lower than the melting temperature(Tm) of the crossbred between described oligonucleotide and/or an oligonucleotide analogs and the homology complementary DNA (DNA crossbred).

Class RNA and class DNA

Class RNA be meant according to the nucleic acid with RNA character or the oligonucleotide analogs of the homology complementary oligonucleotide that comprises at least a inner pseudonucleus thuja acid and/or oligonucleotide analogs nucleic acid analog or oligonucleotide analogs hybridization.Therefore, class RNA nucleic acid analog or oligonucleotide analogs can carry out classification on the function according to the ability of they and oligonucleotide and/or oligonucleotide analogs hybridization difference RNA and DNA.Preferably, the described oligonucleotide analogs that can distinguish RNA and DNA comprises that one or more are positioned at inner pseudonucleus thuja acid intercalator and therefore, comprise the described oligonucleotide analogs of pseudonucleus thuja acid intercalator will be preferably with described class RNA nucleic acid or oligonucleotide analogs hybridization.

The embodiment of class RNA molecule is RNA, 2 '-O-methyl-RNA, LNA, α-L-ribo-LNA, α-L-ribo-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, 2 '-R-RNA, 2 '-OR-RNA and composition thereof.

Similarly, class DNA is meant according to hybridizing nucleic acid analog or the oligonucleotide analogs with DNA character with homology complementary nucleic acid and/or nucleic acid analog.Therefore, class DNA nucleic acid or nucleic acid analog can carry out classification on the function according to the ability of they and oligonucleotide and/or oligonucleotide analogs hybridization difference RNA and DNA.Preferably, can distinguish the described oligonucleotide of RNA and DNA or oligonucleotide analogs and comprise that one or more are positioned at inner pseudonucleus thuja acid intercalator and therefore, comprise that the described oligonucleotide analogs of pseudonucleus thuja acid intercalator will be preferably and described class DNA nucleic acid analog or oligonucleotide analogs hybridization.

The example of class dna molecular is DNA and INA (Christensen, 2002, comprise the embedding nucleic acid of 1-O-(1-pyrenyl methyl) glycerine insert: stablize dsDNA and distinguish DNA and RNA, Nucl.Acids.Res.2002 30:4918-4925).

Cross hybridization

Term cross hybridization comprises the random hybridization between at least two nucleic acid and/or the nucleic acid analog, i.e. cross hybridization also can be expressed as intermolecular hybridization.Therefore term cross hybridization for example can be used for describing nucleic acid probe or nucleic acid analog probe sequence and other nucleotide sequence and/or nucleic acid analog but not the hybridization of its specified target sequence.

Usually, cross hybridization occurs between the complementary non-target sequence of probe and one or more homologies, even they have than probe and complementary target sequence thereof the complementarity of low degree more.This unwanted effect due to probe than the excessive far away and/or rapid annealing kinetic of relative target.Cross hybridization also takes place by the hydrogen bonding between several nuclear base pairs, between the primer during for example PCR reacts, thereby produces the formation of primer dimer and/or the formation of non-specific PCR product.

Especially, the nucleic acid that comprises one or more nucleotide analogs that has a height affinity with the nucleotide analog of same type trends towards forming based on the dipolymer of base pairing or high-grade mixture more.Especially, comprise such as, but not limited to DNA, RNA, 2 '-probe of O-methyl RNA, PNA, HNA, MNA, ANA, LNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, 2 '-R-RNA, 2 '-OR-RNA and composition thereof comprises to other that unitary oligonucleotide analogs of same type backbone monomers has usually and highly hybridizes affinity.Even therefore independent probe molecule has only the complementarity of lower level, they still trend towards hybridization.

Oneself's hybridization

Term oneself hybridization comprises that its amplifying nucleic acid or nucleic acid analog molecule are annealed to himself by folding on himself, thereby produces for example class secondary structure of hairpin structure, i.e. oneself's hybridization also can be defined as intramolecularly hybridization.In great majority were used, it was very important avoiding oneself's hybridization.The generation of described secondary structure can suppress the hybridization with the nucleic acid target sequence of expecting.In great majority were analyzed, this was undesirable, for example when nucleic acid or nucleic acid analog during as the primer in the PCR reaction or as the probe of the fluorophore/quencher mark of exonuclease analysis.In these two kinds of analyses, self-hybridization will suppress with the hybridization of target nucleic acid and exonuclease analysis in the level of other fluorophore quencher also reduce.

Especially, the nucleic acid of the nucleotide analog that comprises one or more and same type with nucleotide analog of height affinity trends towards oneself's hybridization.Especially, comprise probe such as, but not limited to following nucleotide analog have usually to the oneself hybridize higher affinity: DNA, RNA, 2 '-O-methyl RNA, PNA, HNA, MNA, ANA, LNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, 2 '-R-RNA, 2 ' OR-RNA.Even it is complementary that therefore independent probe molecule has only the oneself of lower level, they still trend towards oneself's hybridization.

Melting temperature(Tm)

The thermal separation of two chains that the unwinding of nucleic acid is meant double chain acid molecule.

Melting temperature(Tm) (Tm) expression is with respect to (the not hybridizing) centigradetemperature when there is 50% spiral (hybridization) form in form of curling.

Higher melting temperature(Tm) shows as a kind of stabilized complex and therefore have higher affinity between independent chain.Vice versa, and low melting temperature(Tm) shows as affinity relatively low between the independent chain.Therefore, stronger hydrogen bonding produces higher melting temperature(Tm) between common two chains.

In addition, intercalator disclosed by the invention is embedded into and also may stablizes double-strandednucleic acid between the nuclear base of double-strandednucleic acid and therefore produce higher melting temperature(Tm).

Melting temperature(Tm) depends on the physical/chemical state of surrounding environment in addition.For example melting temperature(Tm) depends on salt concn and pH.

Melting temperature(Tm) may be determined by multiple analysis, for example may determine the formation and the fracture (unwinding) of hybridization by utilizing ultra-violet absorption spectrum.

The backbone monomers unit

The backbone monomers unit of Nucleotide of the present invention or nucleotide analog or intercalator pseudonucleus thuja acid is the part of Nucleotide, and it participates in Nucleotide or nucleotide analog or intercalator pseudonucleus thuja acid and embeds in the main chain of nucleic acid or nucleic acid analog.The present invention can use arbitrary suitable backbone monomers unit.

The backbone monomers unit of intercalator pseudonucleus thuja acid especially of the present invention can be selected from the backbone monomers unit of mentioning below.

The backbone monomers unit comprises part Nucleotide or the nucleotide analog or the intercalator pseudonucleus thuja acid that may merge in oligonucleotide or the oligonucleotide analogs main chain.In addition; the backbone monomers unit can comprise one or more leavings groups; protecting group and/or active group, its can be by any way between synthesis phase or remove after unitary oligonucleotide of described backbone monomers or the oligonucleotide analogs or change synthetic comprising.

Term backbone monomers importantly of the present invention unit only comprises backbone monomers unit own and it does not for example comprise, and the backbone monomers unit is connected to the joint of intercalator.Therefore, intercalator and joint are not the unitary parts of backbone monomers.

Therefore, the backbone monomers unit only comprises atom, and wherein monomer embeds sequence, and described atom is selected from:

A) can form the atom of chemical bond with the backbone monomers unit of adjacency Nucleotide; Perhaps

B) at least two sites are connected to the atom of other atom of backbone monomers unit; Perhaps

C) site be connected to the backbone monomers unit and otherwise the atom that is not connected with other atom more preferably, therefore backbone monomers unit atom is interpreted as when monomer embeds sequence, directly connect the atom of (shortest path) between the main chain phosphor atoms of participation adjacent nucleotide, wherein adjacent nucleotide is naturally occurring Nucleotide.

The backbone monomers unit can be arbitrary suitable backbone monomers unit.In one embodiment of the invention, the backbone monomers unit for example can be selected from and comprise DNA, RNA, PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, the backbone monomers unit of β-D-RNA.

Described below is a large amount of different Nucleotide and the backbone monomers unit of nucleotide analog, and how they to be connected to the nuclear base by joint, and described joint is connected to unitary one or two sites of backbone monomers:

DNA, RNA﹠amp; The example of the oligomer of PNA

Ref.Nielsen .Science such as P.E., 1991,254,1497.

The example of the oligomer of some analogues

Ref.Allart, Chem.Eur.J. such as B., 1999,5,2424-2431.

Ref.Singh.S.K. wait Chem.Commun., 1998,455-456:Koshkin, Tetrahedron such as A.A., 1998,54,3607-3630; Obiks, S.et al.Tetrahedron lett., 1997,38,8735-8738.

The example of the oligomer of some analogues of cyclohexyl-NA (CNA)

???

Ref：Maurinsh，Y.；et?al.Chem.Eur.J.，1999，2139-2150.

Cyclohexenyl-NA (CeNA)

?????

Ref：Wang，J.：et?al.J.Am.Chem.Soc，2000，8595-8602.

(2′-NH)-TNA????????????????????????(3′-NH)-TNA

Ref.：Wu，X.et?al.，Org.Lett.，2002，4，1279-1282

TNA

?????

Ref.：Wu：X.et?al.，Org.Lett.，2002，4，1279-1282

The nucleic acid moiety of α-each analogue of L-ribo-LNA

???

Ref：Rajwanshi，V.K.et?al.Chem.Commun.，1999，1395-1396.

α-L-wood sugar-LNA

?????

Ref：Rajwanshi，V.K?et?al.Angew.Chem.Int.Ed.，2000，1656-1659.

β-D-wood sugar-LNA

????

Ref：Rajwanshi，V.K.et?al.Chem.Commun.，1999，1395-1396.

α-D-ribo-LNA

?????

Ref：Rajwanshi，V.K?et?al.Angew.Chem.Int.Ed.，2000，1656-1659.

[3.2.1]-LNA

?????

Ref：Wang，G.；et?al.Tetrahedron，1999，7707-2724.

Two ring-DNA 6 '-amino-two ring-DNA 5 '-Biao-two ring-DNA

Three ring-DNA α-two ring-DNA, two ring [4.3.0]-DNA

Two ring [3.2.1]-DNA, two ring [3.2.1] acid amides-DNA

Ref：All?of?the?Bicyclo-DNAs?are?reviewed?in?Leumann，C.J.，Bioorg.Med.Chem.，2002，841-854.

β-D-ribopyranose base-NA α-L-pyrans lysol glycosyl-NA

????

Ref：Reck，F.et?al.，Org.Lett.1999，1，1531?????Ref：Reck，F.et?al.，Org.Lett.1999，1，1531

2 '-formula of the oligonucleotide of R-RNA 2 '-modification

Ref：Reviewed?by?Manoharan，M.Biochim.BioPhys.Acta，1999，117-130.

????????????????????????????????????????

Ref：Yamana，K.et?al.，Tetrahedron?Lett.，1991，6347-6350.????Ref：Sayer，J.et?al.，J.Org.Chem.，1991，56，20-29.

From the information that we grasp, the example of synthetic as yet or disclosed modification:

The backbone monomers unit of LNA (nucleic acid of sealing) is a restriction enzyme digestion dna backbone monomeric unit on the space, and it contains the common conformational freedom that the interior bridge of a part has limited the dna backbone monomeric unit.LNA can be arbitrary LNA molecule of describing among the WO 99/14226 (Exiqon), and preferably LNA is selected from the molecule that is described among the WO 99/14226.Preferred LNA of the present invention contains 12 '-O position is connected to the methyl joint of 4 '-C position, yet other LNA ' s such as 2 ' Sauerstoffatom wherein is also included within the scope of the present invention by the LNA ' s that nitrogen or sulphur replace.

The backbone monomers unit of intercalator pseudonucleus thuja acid of the present invention preferably had following formula before embedding oligonucleotide and/or nucleotide analog:

Wherein n=1 to 6, preferably n=2 to 6, more preferably n=3 to 6, more preferably n=2 to 5, more preferably n=3 to 5, more preferably n=3 to 4.

R ₁Be the phosphorus atom that trivalent or pentavalent replace, preferably R ₁Be

Or

Wherein

R ₂Be selected from the atom that can form at least two keys discriminably, described atom optionally replaces respectively, preferably R ₂Be selected from the O that selectivity replaces respectively respectively, S, N, C, P.Term " difference " is meant R ₂Can be illustrated in the same molecular is one, the group that two or more are different.Two R ₂Between chemical bond can be saturated or undersaturated or ring system a part or and the combination.

Each R ₂Can replace with arbitrary suitable substituents respectively, such as being selected from H, low alkyl group, C2-6 thiazolinyl, C6-10 aryl, C7-11 arylmethyl, the non-epoxy methyl of C2-7, C3-8 alkoxy carbonyl oxygen methyl, C7-11 aroyl oxygen methyl, C3-8 S-acyl group-2-thio-ethyl;

" alkyl " is meant the saturated aliphatic hydrocarbon that selectivity replaces, and comprises straight chain, side chain and cycloalkyl.Preferably, alkyl has 1 to 25 carbon and contains and is no more than 20 heteroatoms.More preferably, it is the low alkyl group of from 1 to 12 carbon atom, more preferably 1 to 6 carbon, more preferably 1 to 4 carbon.Heteroatoms preferably is selected from nitrogen, sulphur, phosphorus and oxygen.

" thiazolinyl " is meant that an optional hydrocarbon polymer that replaces comprises at least one two key, comprises straight chain, side chain and ring-type thiazolinyl, and all can choose replacement wantonly.Preferably, thiazolinyl has 2 to 25 carbon and comprises and is no more than 20 heteroatoms.More preferably, it is the low-grade alkenyl of from 2 to 12 carbon atoms, more preferably 2 to 4 carbon atoms.Preferably heteroatoms is selected from nitrogen, sulphur, phosphorus and oxygen.

" alkynyl " is meant an optional unsaturated hydrocarbons that replaces, and comprises at least one triple bond, comprises straight chain, side chain and ring-type alkynyl, and all can choose replacement wantonly.Preferably, alkynyl has 2 to 25 carbon and comprises and is no more than 20 heteroatoms.More preferably, it is the low-grade alkynyl of from 2 to 12 carbon atoms, more preferably 2 to 4 carbon atoms.Preferably heteroatoms is selected from nitrogen, sulphur, phosphorus and oxygen.

" aryl " be meant all at least one rings have the conjugated pi electron system the aryl of optional replacement, and comprise isocyclic aryl, heterocyclic aryl, biaryl and triaryl.The example of aryl substituent comprises amino, carboxyl, hydroxyl, alkoxyl group, nitro, alkylsulfonyl, halogen, mercaptan and the aryloxy of alkyl, thiazolinyl, alkynyl, aryl, amino, replacement.

" isocyclic aryl " is meant an aryl, and wherein all atoms on the aromatic nucleus all are carbon atom.Carbon atom is chosen substituted aryl wantonly as mentioned above.Preferably, isocyclic aryl is an optional phenyl that replaces.

" heterocyclic aryl " is meant that one has the aryl of 1 to 3 heteroatoms as the annular atoms in the aromatic nucleus, and remaining annular atoms is a carbon atom.The heteroatoms that is fit to comprises oxygen, sulphur and nitrogen.The example of heterocyclic aryl comprises furyl, thienyl, and pyridyl, pyrryl, N-reduces alkyl pyrrolo-, pyrimidyl, pyrazinyl and imidazolyl.As above the described heterocyclic aryl of aryl is optionally substituted.

At two or more R ₂On substituting group can be connected to form a ring system in addition, such as arbitrary ring system as defined above.

R preferably ₂Be selected from H, methyl, R with an atom or ₄, hydroxyl, halogen and amino group replace, R more preferably ₂With an atom or be selected from hydrogen, methyl, R ₄Group replace.

More preferably, R ₂Be selected from O, S, NH, N (Me), N (R respectively ₄), C (R ₄) ₂, CH (R ₄) or CH ₂, R wherein ₄The following definition,

R ₃=methyl, β-cyanoethyl, right-the nitro phenelyl, neighbour-chloro-phenyl-or right-chloro-phenyl-.

R ₄=low alkyl group, preferably such as the low alkyl group of methyl, ethyl or sec.-propyl, perhaps such as morpholino, tetramethyleneimine generation or 2,2,6, the heterocycle in 6-tetramethylpyrrolidi-e generation, wherein low alkyl group is defined as C ₁-C ₆, such as C ₁-C ₄

R ₅=alkyl, alkoxyl group, aryl or H, collateral condition is when X2=O-, R ₅Be hydrogen, preferably R ₅Be selected from low alkyl group, lower alkoxy, aryloxy.In a preferred embodiment, aryloxy is selected from phenyl, naphthyl or pyridine.

R ₆Be a protecting group, be selected from arbitrary suitable protecting group.R preferably ₆Be selected from and comprise trityl; single methoxy trityl; 2-chloro trityl; 1,1,1; 2-tetrachloro-2; two (right-the p-methoxy-phenyl)-ethane (DATE) of 2-, 9-phenyl yanthine-9-base (pixyl) and 9-(right-p-methoxy-phenyl) xanthine-9-base (MOX) or other are at " experimental technique of current nucleic acid chemistry (Current Protocols In Nucleic Acid Chemistry) " first roll of Beaucage Wiley. etc., in the protecting group mentioned.More preferably, protecting group may be selected from mono methoxy trityl and dimethoxytrityl.Most preferably, protecting group can be 4,4 '-dimethoxytrityl (DMT).

R ₉Be selected from optional O, S N, the preferably R that replaces ₉Be selected from O, S, NH, N (Me).

R ₁₀Be selected from optional O, S, N, the C that replaces.

X ₁=Cl, Br, I or N (R ₄) ₂

X ₂=Cl, Br, I, N (R ₄) ₂, perhaps O ^-

According to substituting group as mentioned above, the backbone monomers unit can be acyclic or part ring system.

In a preferred embodiment of the invention, the backbone monomers unit of intercalator pseudonucleus thuja acid is selected from acyclic backbone monomers unit.Acyclic is meant and comprises arbitrary backbone monomers unit, and it does not comprise a ring structure, and for example the backbone monomers unit does not preferably comprise ribose or ribodesose group.

Especially, preferably the backbone monomers unit of intercalator pseudonucleus thuja acid is an acyclic backbone monomers unit, and it can stablize protrusion insert (referring to following explanation).

In another preferred embodiment, the backbone monomers unit of intercalator pseudonucleus thuja acid of the present invention may be selected from and comprise that at least one is selected from the backbone monomers unit of trivalent and pentavalent phosphorus atom such as the chemical group of pentavalent phosphorus atom.More preferably, the unitary phosphoric acid ester atom of intercalator pseudonucleus thuja acid backbone monomers of the present invention may be selected from and comprise that at least one is selected from the backbone monomers unit of the chemical group of phosphoric acid ester, phosphodiester, phosphoramidate phosphoramidit.

Especially, intercalator pseudonucleus thuja acid backbone monomers preferably of the present invention unit is selected from the acyclic backbone monomers unit of the chemical group that comprises that at least one is selected from phosphoric acid ester, phosphodiester, phosphoramidate and phosphoramidit.

The preferred backbone monomers unit that comprises the chemical group that at least one is selected from phosphoric acid ester, phosphoric acid ester, phosphodiester and phosphoramidit is the backbone monomers unit, wherein when the backbone monomers unit is embedded in the nucleic acid main chain, it is long that the distance of at least one phosphorus atom from least one phosphorus atom to contiguous Nucleotide does not comprise that the words of phosphor atoms are at least 6 atoms, for example 2 such as 3, for example 4, such as 5, for example 6 atoms are long.

Measure distance as mentioned above as direct chemical key (that is shortest path).

Preferably the backbone monomers unit can so that at the most 5 atoms the mode that the unitary phosphorus atom of intercalator pseudonucleus thuja acid backbone monomers and nearest neighbour phosphorus atom separate is embedded in nucleic acid or the nucleic acid analog, do not comprise in both cases that more preferably 5 atoms of phosphorus atom self are separated unitary phosphorus atom of intercalator pseudonucleus thuja acid backbone monomers and nearest neighbour phosphorus atom.

Preferably, the backbone monomers unit can so that at the most 4 atoms the mode that the unitary phosphorus atom of intercalator pseudonucleus thuja acid backbone monomers and nearest neighbour phosphorus atom separate is embedded in nucleic acid or the nucleic acid analog, do not comprise in both cases that more preferably 4 atoms of phosphorus atom self are separated unitary phosphorus atom of intercalator pseudonucleus thuja acid backbone monomers and nearest neighbour phosphorus atom.

In the present invention's one especially preferred embodiment, intercalator pseudonucleus thuja acid comprises that one contains the backbone monomers unit of phosphoramidit, and more preferably the backbone monomers unit comprises a trivalent phosphoramidit.

The trivalent phosphoramidits that is fit to is trivalent phosphoramidit, and it can be embedded in the main chain of nucleic acid and/or nucleic acid analog.Usually, amidit group self may not be embedded in the main chain of nucleic acid, yet amidit group or part amidit group can be used as leavings group and/or protecting group.Yet preferably the backbone monomers unit comprises a phosphoramidit group, because this group can promote the backbone monomers unit to embed in the nucleic acid main chain.

Preferably the acyclic backbone monomers may be selected from a following formula:

R wherein ₁, R ₂And R ₆As defined above.

More preferably, acyclic backbone monomers unit may be selected from following group:

R wherein ₁, R ₂And R ₆As defined above, and R ₇=N, perhaps CH.

Be that the backbone monomers unit is from 1 below) specific examples of open numbering, wherein R ₁And R ₆As defined above, and R ₈May be the optional R that replaces ₄Perhaps H.

Me represents methyl

More preferably, the backbone monomers unit that comprises optional protecting group can be selected from the I of containing as described below) to XLIV) group of structure:

Most preferred for being selected from the backbone monomers unit of following groups:

Preferably, acyclic backbone monomers unit may be selected from contain as described below a) to g) group of structure:

According to the present invention, the backbone monomers unit that embeds the intercalator pseudonucleus thuja acid of oligonucleotide or oligonucleotide analogs can comprise a phosphodiester bond.In addition, according to the present invention, the backbone monomers unit of intercalator pseudonucleus thuja acid can comprise a pentavalent phosphoramidate.Preferably, according to the present invention, the backbone monomers unit of intercalator pseudonucleus thuja acid is for comprising the acyclic backbone monomers unit of a pentavalent phosphoramidate.

Leavings group

Backbone monomers of the present invention unit can comprise one or more leavings group.Leavings group is a chemical group, when intercalator pseudonucleus thuja acid or Nucleotide are monomer, it is part backbone monomers unit, in case but intercalator pseudonucleus thuja acid or Nucleotide have embedded oligonucleotide or the described leavings group of oligonucleotide analogs just no longer is present in the molecule.

The characteristic of leavings group depends on the backbone monomers unit.For example, when the backbone monomers unit was a phosphorescence amidit, leavings group for example can be a Diisopropylamine group.Usually, when the backbone monomers unit is a phosphorescent substance amidit, leavings group for example is attached to phosphorus atom with the form of Diisopropylamine, and described leavings group is removed after phosphorus atom is connected to nucleophilic group, and remaining bound phosphate groups or remainder can become part nucleic acid or nucleic acid analog main chain.

Active group

Backbone monomers of the present invention unit can comprise one in addition can carry out the active group that chemical reaction forms nucleic acid or nucleic acid analog with another Nucleotide or oligonucleotide or nucleic acid or nucleic acid analog, and described nucleic acid or nucleic acid analog are than the long Nucleotide of reactant.

Therefore, when Nucleotide exists with free form, when promptly not embedding nucleic acid, they can comprise one can with another Nucleotide or nucleic acid or nucleic acid analog reactive activity group.

In a preferred embodiment of the invention, described active group can be protected by blocking group.Before described chemical reaction, can remove described protecting group.Therefore protecting group just is not a new part that forms nucleic acid or nucleic acid analog.

The example of active group is a nucleophilic group, such as DNA or the unitary 5 '-hydroxyl of RNA backbone monomers.

Protecting group

Backbone monomers of the present invention unit also may comprise a protecting group that can be removed, and wherein removing of protecting group can be carried out chemical reaction between intercalator pseudonucleus thuja acid and Nucleotide or nucleotide analog or another intercalator pseudonucleus thuja acid.

Especially; nucleotide monomer or nucleotide analog monomer or intercalator pseudonucleus thuja acid monomer can comprise protecting group; in case Nucleotide or nucleotide analog or intercalator pseudonucleus thuja acid have embedded nucleic acid or nucleic acid analog, it no longer is present in the molecule.

In addition; after Nucleotide or nucleotide analog or the embedding of intercalator pseudonucleus thuja acid; the backbone monomers unit can comprise the protecting group that may be present among oligonucleotide or the oligonucleotide analogs; but backbone monomers cell protection base may no longer exist after other Nucleotide or nucleotide analog are introduced oligonucleotide or oligonucleotide analogs, perhaps removes behind synthetic whole oligonucleotide or oligonucleotide analogs.

Protecting group may be removed by the known multiple suitable technique of person skilled in the art, yet preferably, protecting group may be removed by the processing that is selected from group acid treatment, phenylmercaptan processing and alkaline purification.

The present invention can be used to protect backbone monomers unitary 5 ' preferred protecting group terminal or 5 ' terminal analogue may be selected from trityl; single methoxy trityl; 2-chlorine trityl; 1; 1; 1; 2-tetrachloro-2; two (right-the p-methoxy-phenyl)-ethan (DATE) of 2-, the protecting group that 9-phenyl yanthine-9-base (pixyl) and 9-(right-p-methoxy-phenyl) xanthine-9-base (MOX) or other are mentioned in " experimental technique of current nucleic acid chemistry (Current Protocols InNucleic Acid Chemistry) " first roll of Beaucage Wiley. etc.More preferably, protecting group may be selected from mono methoxy trityl and dimethoxytrityl.Most preferably, protecting group can be 4,4 '-dimethoxytrityl (DMT).

4,4 '-dimethoxytrityl (DMT) group may be removed by acid treatment, for example by the CH of brief incubation (being enough in 30 to 60 seconds) at 3% trichoroacetic acid(TCA) or 3% dichloro acetic acid ₂Cl ₂In the solution.

Can protect the preferred protecting group of unitary phosphoric acid ester of backbone monomers or phosphoramidit group can for example be selected from methyl and 2-cyanoethyl.The methyl protecting group may for example be passed through with phenylmercaptan or 2-carbamyl-2-vinyl cyanide-1, and 1-disodium thiolate is handled and removed.2-cyanoethyl-group may for example be used concentrated liquor by alkaline purification, 1: 1 mixture of methylamine water and concentrated liquor or remove with ammonia.

Intercalator

Term intercalator of the present invention comprises the arbitrary molecular moiety that contains at least one generally planar conjugate system, and described conjugate system can be piled up altogether with the nuclear base of nucleic acid.Preferably, intercalator of the present invention is made up of at least one generally planar conjugate system basically, and described conjugate system can be piled up altogether with the nuclear base of nucleic acid or nucleic acid analog.

Preferably, intercalator comprises the group that is selected from polyaromates and heteropolyaromates, and more preferably intercalator is made up of polyaromate or heteropolyaromate basically.Most preferably, intercalator is selected from polyaromates and heteropolyaromates.

Polyaromates of the present invention or heteropolyaromates may be made up of the ring of arbitrary suitable quantity, such as 1, and for example 2, such as 3, for example 4, such as 5, for example 6, such as 7, for example 8, such as surpassing 8.In addition, polyaromates or heteropolyaromates can replace with one or more group that is selected from hydroxyl, bromine, fluorine, chlorine, iodine, sulfydryl, sulphur, cyano group, alkyl sulfide, heterocycle, aryl, heteroaryl, carboxyl, carbalkoxy, alkyl, thiazolinyl, alkynyl, nitro, amino, alkoxyl group and amide group.

In embodiment preferred of the present invention, intercalator can be selected from can fluorescigenic polyaromates and heteropolyaromates.

In another preferred embodiment of the present invention, intercalator may be selected from and can form excimer, exciplex, the polyaromates and the heteropolyaromates of fluorescence resonance energy transmission (FRET) or charge-transfer complex.

Therefore, intercalator preferably is selected from phenanthroline; azophenlyene; phenanthridines; anthraquinone; pyrene; anthracene; naphthenic hydrocarbon; luxuriant and rich with fragrance; picene; chrysene; naphtacene; dihydroketoacridine; benzanthrene; the 1-2-toluylene; the oxalo pyrido-carbazole; phenylazide; porphyrin; psoralene and arbitraryly be selected from hydroxyl with one or more; bromine; fluorine; chlorine; iodine; sulfydryl; sulphur; cyano group; alkyl sulfide; heterocycle; aryl; heteroaryl; carboxyl; carbalkoxy; alkyl; thiazolinyl; alkynyl; nitro; amino; the above-mentioned intercalator that the group of alkoxyl group and/or amide group replaces.

Preferably, intercalator be selected from comprise phenanthroline, azophenlyene, phenanthridines, anthraquinone, pyrene, anthracene, naphthenic hydrocarbon, phenanthrene, picene, bend, the group of tetracene (naphtacene), dihydroketoacridine, benzanthrene, 1-2-toluylene, oxalo pyrido-carbazole, phenylazide, porphyrin and psoralene.

More preferably, intercalator may be selected from the group of the intercalator that comprises one or more structures as follows:

Daunorubicin I

Adriamycin II naphthols benzoquinones

Psoralen IV phenanthroline 1, the 3-dithiole,

2-(1,3-dithiol-2-subunit)-

?????????

Trimethylammonium psoralen fluorescein derivative

The acridine derivatives acridine

Acridine pyrene naphthalene

The luxuriant and rich with fragrance benzophenanthrene of Antracene

6H-indyl [2,3-b] quinoxaline quinoline 1H-indenes benzothiazole

7,8,9,10-tetrahydro benzo [a] pyrene 9,10-amerantrone 1,10-phenanthroline

?? ?????????????

The 9bH-Phenalene pyrido [3 ', 2 ': 4 ' 5] thieno-[3,2-d] pyrimidine perylenes-4 (1H)-ketone, 7, the 9-dimethyl-

6H-pyrido [4,3-b] carbazone, phenanthridines, 3,8-diamino-5-ethyl-6-phenyl-

5, the 11-dimethyl-

1,2-naphthalenedione 1H-benzoglyoxaline 1H-indoles 1,4-naphthalenedione

Dibenzo [a, g] quinolizine,

2,3,10,11-tetramethoxy-8-methyl-

Phenanthridines, 3,8-diamino-[3-(diethylmethyl 1H-benzene [de] isoquinoline 99.9-1,3 (2H)-diketone amino) propyl group]-6-phenyl-

Naphthalene, 1,2-dimethoxy-two pyrido [3,2-a:2 ' 3 '-c] azophenlyene

Quinoline, 4-[(3-ethyl-2 (3H)-benzacridine, 6-amino-3,10-two

Bing oxazinyl subunit) methyl]-1-methyl-hydrogen-3-imino--10-methyl-

Acridine, 9-amino-10-methyl-

???????????????????

Quinoline, 1-methyl-4-[(3-methyl-2 (1,3,6,8 (2H, 7H)-pyrene tetraketone 3H)-the benzothiazole subunit) methyl]-

????????????

???????????

Benzo [Imn] [3,8] phenanthroline-1,3,6 2H-1-chromen-2-one xanthenes, the 9-phenyl-, 8 (2H, 7H)-tetraketone

The Ellipticine pyrido [3 ', 2 ': 4,5] thiophene Fulvalene fen [3,2-d] pyrimidines-4 (1H)-ketone also

DAPI

And derivative.

More preferably, intercalator may be selected from and contain above-mentioned V, XII, XIV, XV, XVII, XXIII, XXVI, XXVIII, XLVII, the intercalator and the derivative thereof of the intercalator structure of one of LI and LLI.

Most preferably, intercalator is selected from above-mentioned XII, XIV, XVII, XXIII, the intercalator structure of LI.

The pyrene anthracene

9,10-amerantrone 6H-indoles [2,3-b] quinoxaline

And derivative.

The above-mentioned example of enumerating can not be interpreted as by any way and limit, only provide the example as the possible structure of intercalator.In addition, replacing one or more chemical group on every kind of intercalator is also included within the scope of the present invention with the structure that obtains to modify.

The intercalator part of intercalator pseudonucleus thuja acid is connected to backbone units by joint.When leaving main chain to embedded part along joint, joint and intercalator connect and are defined as the joint atom and belong to key between first atom of conjugate system, when described oligonucleotide or oligonucleotide analogs and comprise oligonucleotide analogs when hybridization of described intercalator pseudonucleus thuja acid, described conjugate system can be piled up altogether with the nuclear base of described oligonucleotide or oligonucleotide analogs chain.

In one embodiment of the invention, joint can comprise that a conjugation system and intercalator can comprise another conjugate system.In this case, the joint conjugate system can not be piled up altogether with the nuclear base of described opposite oligonucleotide or oligonucleotide analogs chain.

Joint

Intercalator pseudonucleus thuja acid of the present invention be one to connect the part of the backbone monomers of intercalator and described intercalator pseudonucleus thuja acid.Joint can comprise the key between one or more atom or the atom.

The definition of main chain and embedded part by above-mentioned definition, joint are the shortest paths that connects main chain and intercalator.If intercalator is directly connected to main chain, joint is exactly a chemical bond.

Joint generally includes an atomchain or monatomic side chain.Chain can be saturated with undersaturated.Joint also can be the ring texture that has or do not have conjugated link(age).

For example joint can comprise a m atomchain that is selected from C, O, S, N, P, Se, Si, Ge, Sn and Pb, and its medium-chain one end is connected to another end of the chain of intercalator and is connected to the backbone monomers unit.

In some embodiments, the total length of the intercalator of joint and intercalator pseudonucleus thuja acid of the present invention preferably between 8 and 13 (referring to following).Therefore, the selection of m should depend on the size of the intercalator of concrete intercalator pseudonucleus thuja acid.

That is, hour m should be relatively large when intercalator, and when intercalator was big, m should be less relatively.Yet at m many times will be from 1 to 7 integral body, such as from 1-6, such as from 1-5, such as from 1-4.Joint may be that unsaturated chain or another comprise the system of conjugated link(age) as mentioned above.For example joint can comprise cyclic conjugated structure.Preferably, when joint was saturated chain, m was from 1 to 4.

When intercalator was pyrene, m was preferably from 1 to 7 integer, such as from 1-6, and such as from 1-5, such as from 1-4, more preferably from 1 to 4, more preferably from 1 to 3, most preferably m is 2 or 3.

When intercalator had the structure of following formula, preferably m was from 2 to 6, more preferably was 2.

Fitting chain can replace with one or more atom that is selected from C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.

In one embodiment, joint is an azepine alkyl, oxa alkyl, thia alkyl or alkyl chain.For example fitting chain can be selected from the alkyl chain of the atom replacement of C, H, O, S, N, P, Se, Si, Ge, Sn and Pb with one or more.In an embodiment preferred, joint comprises a ramose alkyl chain not, and its medium-chain one end is connected to intercalator, another end of the chain be connected to the backbone monomers unit and wherein each C replace with 2H.More preferably, described unbranched alkyl chain from 1 to 5 atom is long, and is long such as from 1 to 4 atom, long such as from 1 to 3 atom, long such as from 2 to 3 atoms.

In another embodiment of the present invention, joint is one to comprise the ring texture of the atom that is selected from C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.The fitting chain this ring texture that can replace for example for the atom that is selected from C, H, O, S, N, P, Se, Si, Ge, Sn and Pb with one or more.

In another embodiment, joint comprises from 1-6 C atom, from 0-3 each O, S, N atom.More preferably, joint comprises from 1-6 C atom, from 0-1 each O, S, N atom.

In a preferred embodiment, joint comprises the atomchain of optional C, O, S and the N that replaces.Preferably described chain should comprise 3 atoms at the most, comprises that therefore from 0 to 3 is selected from optional C, the O that replaces, the atom of S, N respectively.

In an embodiment preferred, joint comprises a C, N, S and O atomchain, and its medium-chain one end is connected to intercalator, and another end of the chain is connected to the backbone monomers unit.

Preferably this chain comprises a joint that shows below, and most preferably joint comprises a molecule that shows below:

CH ₂????H ₂C-CH ₂ O-CH ₂

I???????????II??????????III???????????IV????????V????????VI???????????VII

O?? HC＝CH C≡C

VIII????IX??????????X??????XI????????????XII???????XIII???????XIV??XV??????XVI

S-CH ₂

S

XVII???XVIII?????????XIX??????????XX???????XXI????????XXII????????XXIII???XXIV

HN????NH-CH ₂

XXV??????????????XXVI????????XXVII????XXVIII?????XXIX?????XXX??????????XXXI

XXXII??????????XXXIII?????XXXIV??????????XXXV?????????XXXVI???????????XXXVII????????XXXVIII

HN????NH-CH ₂

XXXIX?????XL??????XLI??????????XLII???????????XLIII?????XLIV????????XLV?????????XLVI

XLVII????????????XLVIII????????XLIX?????????????L???????????????LI?????????????LII

In an embodiment preferred, chain comprises a joint that shows below, and more preferably joint comprises a molecule that shows below:

H ₂C-CH ₂

II

In a preferred embodiment, chain comprises the joint that one of shows below, and more preferably joint comprises a molecule that shows below:

H ₂C-CH ₂

II

Joint is included in as defined above Y in the intercalator pseudonucleus thuja acid X-Y-Q formula, so X and Q are not the integral parts of joint.

Intercalator pseudonucleus thuja acid

Intercalator pseudonucleus thuja acid of the present invention preferably has formula,

X-Y-Q

Wherein X one can embed the backbone monomers unit of nucleic acid or nucleic acid analog main chain,

Q one comprises the intercalator of the conjugate system that at least one is generally planar, and the described conjugate system of nuclear base can be piled up altogether with the nuclear base of nucleic acid; And

Y is the shank of a connection described backbone monomers unit and described intercalator; And

Wherein the total length of Q and Y at 7 in the scope of 20 ,

Collateral condition is when intercalator is pyrene, and the total length of Q and Y is in 9A arrives the scope of 13A.

In addition, in the present invention's one preferred embodiment, intercalator comprises a backbone monomers unit, wherein said backbone monomers unit can so that at the most 4 atoms mode of separating 2 phosphorus atom of main chain embed the phosphoric acid ester main chain of nucleic acid or nucleic acid analog, described phosphorus atom is the main chain of close intercalator.

Intercalator pseudonucleus thuja acid does not preferably comprise can form the hydrogen-bonded nuclear base of Wo Sen-Ke Like.Therefore intercalator pseudonucleus thuja acid of the present invention preferably can not Watson-Crick base pairing.

Preferably, the total length of Q and Y at 7 in the scope of 20 , more preferably, from 8 to 15A, more preferably from 8 to 13 , more preferably from 8.4 to 12 , most preferably from 8.59 to 10 or from 8.4 to 10.5 .

When intercalator is pyrene, the total length of Q and Y preferably at 8 in the scope of 13 , such as from 9 to 13 , more preferably from 9.05 to 11 , such as from 9.0 to 11 , from 9.05 to 10 more preferably, such as from 9,0 to 10 , most preferably about 9.8 .

The total length of joint (Y) and intercalator (Q) should be determined to the distance at the non-hydrogen atom center of the generally planar conjugate system of intercalator from the center of joint non-hydrogen atom by definite, described joint and intercalator distance farthest, described conjugate system and backbone monomers cell distance are farthest.Preferably, distance should be the ultimate range that bond angle and normal chemical law do not interrupt or distort by any way.

Distance preferably should have the structure of the free embedding pseudonucleus thuja acid of minimum conformational energy level by calculating, determining then may be from the non-hydrogen atom center of joint to the ultimate range at the non-hydrogen atom center of the generally planar conjugate system of intercalator, described joint and intercalator distance are farthest, if it is not crooked, extend or during the more than simple rotation of chemical bonding structure that opposite twists can rotate freely, conjugate system and backbone monomers cell distance (for example do not participate in the two strands or the chemical bond of ring texture) farthest.

Preferably the structure that energy is suitable is by the structure that starts anew or field of force calculating is found.

More preferably, distance should be determined by the method that comprises the following steps:

A) structure of purpose intercalator pseudonucleus thuja acid utilizes program ChemWindow  6.0 (BioRad) to obtain by computer; And

B) structure is changed over to computer program SymApps ^TM(BioRad); And

C) comprise that the chemical bond length of intercalator pseudonucleus thuja acid of calculating and the 3-dimension structure of bond angle utilize computer program SymApps ^TM(BioRad) calculate; And

D) change 3 dimension structures over to computer program Ras Win MolecularGraphic Ver.2.6-ucb; And

E) utilize RasWin Molecular Graphic Ver.2.6-ucb rotation chemical bond to obtain ultimate range (as defined above); And

F) determine distance.

For example when intercalator pseudonucleus thuja acid has following array structure,

The total length of Q and Y is determined to the slant range of the distance of the former subcenter at B place from the former subcenter of A by measuring, is 9,79 in above-mentioned example.

In another example, intercalator pseudonucleus thuja acid has following array structure:

Is 8.71 from the former subcenter in A site to the collinear Q of the former subcenter mensuration in B site and the total length of Y.

Following public is the straight line measured length of the intercalator pseudonucleus thuja acid of a preferred series:

Intercalator pseudonucleus thuja acid of the present invention may be arbitrary above-mentioned backbone monomers unit, the combination of joint and intercalator.

In one embodiment of the invention, intercalator pseudonucleus thuja acid be selected from have here show 1) to the intercalator pseudonucleus thuja acid of 9 structures:

Wherein DMT and (CH ₂CH ₂CN) play protecting group.

In a preferred embodiment of the invention, intercalator pseudonucleus thuja acid is selected from the phosphoramidits of 1-(4,4 '-dimethoxy triphenyl methoxyl group)-3-pyrene methoxyl group-2-propyl alcohol.More preferably, intercalator pseudonucleus thuja acid is selected from (S)-1-(4,4 '-the dimethoxytrityl methoxyl group)-phosphoramidit of the phosphoramidit of 3-pyrene methoxyl group-2-propyl alcohol and (R)-1-(4,4 '-dimethoxytrityl methoxyl group)-3-pyrene methoxyl group-2-propyl alcohol.

The preparation of intercalator pseudonucleus thuja acid

Intercalator pseudonucleus thuja acid of the present invention can be synthetic by the method that is fit to.

A1) provide and comprise intercalator and a compound that is connected to the shank of active group randomly, described intercalator comprises at least a conjugate system of planar basically that can pile up altogether with the nuclear base of nucleic acid; With

C1) thus with the reaction of described intercalator and described tab precursor and obtain one intercalator-joint; With

D1) provide a backbone monomers precursor unit that comprises at least two active groups and randomly comprise a shank, described two active groups can randomly be protected respectively and/or shelter); With

E1) with described intercalator-joint and described backbone monomers precursors reaction and obtain one intercalator-joint-backbone monomers precursor;

Perhaps

A2) provide a backbone monomers precursor unit that comprises at least two active groups and randomly comprise a shank, described two active groups can randomly be protected respectively and/or shelter); And

B2) provide a tab precursor molecule that comprises at least two active groups, described two active groups can randomly be protected respectively; And

C2) thus with described monomer precursor unit with the reaction of described tab precursor and obtain one main chain-joint; And

D2) provide one to comprise intercalator and a compound that is connected to the shank of active group randomly, described intercalator comprises at least a flat basically planar conjugate system, and described planar conjugate system can pile up altogether with the nuclear base of nucleic acid; And

E2) with described intercalator and described main chain-joint reaction and obtain one intercalator-joint-backbone monomers precursor;

Perhaps

A3) provide one to comprise intercalator and the compound that is connected to the shank of active group, described intercalator comprises at least a conjugate system of planar basically, and it can be piled up altogether with the nuclear base of nucleic acid; With

C3) with the reaction of described intercalator shank and described backbone monomers precursor joint and obtain one intercalator-joint-backbone monomers precursor; And

J) randomly protect and/or go to protect described intercalator-joint-backbone monomers precursor; With

K) providing one, comprise can be with two pseudonucleus thuja acids, the phosphorescent substance of the compound that Nucleotide and/or nucleotide analog couple together; With

I) with described P contained compound and described intercalator-joint-backbone monomers precursors reaction; With

M) obtain an intercalator pseudonucleus thuja acid

Preferably, the selection of intercalator active group is satisfied it and can be reacted with the joint active group.Therefore, if the joint active group is a nucleophile, so preferably the intercalator active group is an electrophilic reagent, and more preferably electrophilic reagent is selected from alkylhalide group, methylsulfonyl oxyalkyl and tolylsulfonyl oxyalkyl.More preferably the intercalator active group is a chloromethyl.Perhaps, the intercalator active group can be a nucleophilic group, for example comprises hydroxyl, mercaptan, Selam, amine or its mixture.

Preferably, ring-type or acyclic alkanes can be polysubstituted alkane or the alkoxyl groups that contains at least three joint active groups.More preferably, polysubstituted alkane can comprise three nucleophilic groups, such as, but not limited to alkane triole, amino alkane glycol or mercapto alkane glycol.Preferably polysubstituted alkane comprises a nucleophilic group, and it is than other the reactivity that has more, and 2 in the nucleophilic group can protect by protecting group in addition.More preferably, ring-type or acyclic alkanes are 2,2-dimethyl-4-methyl hydroxyl-1, and 3-dioxalan, more preferably alkane is D-α, β-isopropylidene glycerine.

Preferably, the joint active group can with intercalator active group react with, for example the joint active group can be a nucleophilic group, for example is selected from hydroxyl, mercaptan, Selam and amine, is preferably hydroxyl.Perhaps, the joint active group can be an electrophilic group, for example is selected from halogen, triflates, methanesulfonates and tosylate.In a preferred embodiment, at least 2 joint active groups can be protected by protecting group.

Described method may further include one or more active group step of connecting with a protecting group and intercalator-precursor monomer.For example the DMT group can be connected to halogen by providing one, adds such as the DMT of Cl and with DMT-Cl and the reaction of at least one joint active group.Therefore, preferably at least one joint active group will be suitable and one be protected.If this step with contain the phosphorus reagent react before carry out, phosphorus reagent can be only and a joint active group reaction so.

Phosphorus reagent for example can be phosphoramidit, for example NC (CH ₂) ₂OP (Npr ⁱ ₂) ₂Perhaps NC (CH ₂) ₂OP (Npr ⁱ) Cl.Preferably phosphorus reagent can be at alkali, such as N (et) ₃, N (' pr) ₂Et and CH ₂Cl ₂Under the condition that exists with the intercalator precursors reaction.

A specificity limiting examples of intercalator pseudonucleus thuja acid synthetic method of the present invention is summarised among embodiment 1 and Fig. 1.

In case determined the proper sequence of oligonucleotide or oligonucleotide analogs, preferably utilize can commercially available acquisition the method and apparatus chemosynthesis: for example the solid phase phosphoramidite method can be used for producing comprise intercalator pseudonucleus thuja acid than short oligonucleotide or oligonucleotide analogs.

The protecting group mentioned in can " experimental technique of current nucleic acid chemistry (Current Protocols In Nucleic AcidChemistry) " first roll of oligonucleotide or oligonucleotide analogs for example by arbitrary BeaucageWiley of being described in etc.

An object of the present invention is to provide synthetic the comprise oligonucleotide of at least one intercalator pseudonucleus thuja acid or the method for oligonucleotide analogs in addition, wherein synthetic can comprising the steps:

C) add cap randomly for unreacted described upholder bonded oligonucleotide; And

D) randomly further described oligonucleotide analogs is prolonged to the oligonucleotide analogs of expecting sequence by increasing one or more Nucleotide, nucleotide analog or intercalator pseudonucleus thuja acid; And

E) described oligonucleotide analogs and described solid support are separated; With

F) thus obtain to comprise the described oligonucleotide analogs of at least a intercalator pseudonucleus thuja acid.

In one embodiment of the invention, synthetic can comprising the following steps:

A. the intercalator pseudonucleus thuja acid that invention is comprised active group contacts with upholder bonded oligonucleotide that is increasing or oligonucleotide analogs chain, and described active group is by sour unsettled blocking group protection; With

B. with described intercalator pseudonucleus thuja acid and described upholder bonded oligonucleotide or oligonucleotide analogs reaction; And

C. the product from the upholder rinses out excess reactant; And

D. randomly unreacted described upholder bonded oligonucleotide is added cap; With

E. oxidation phosphorous acid ester product is to phosphate product; And

F. the product from the upholder rinses out excess reactant; And

G. randomly unreacted described upholder bonded oligonucleotide is added cap; With

H. repeating step a)-g) embeds up to the intercalator pseudonucleus thuja acid of desired amount; And

I. randomly prolong the described upholder bonded oligonucleotide that comprises at least one intercalator pseudonucleus thuja acid; And

J. repeating step a-i randomly)

K. oligonucleotide analogs and solid support are separated and remove alkali labile protecting group in the alkaline medium; And

L. purifying comprises the oligonucleotide analogs of sour unstable protection base; And

M. remove the unsettled protecting group of disacidify with acidic medium; And

N. obtain the oligonucleotide analogs that a terminal pseudonucleus thuja acid that comprises at least one intercalator pseudonucleus thuja acid is modified

In another embodiment of the present invention, synthetic can comprising the steps:

A) intercalator pseudonucleus thuja acid of the present invention is contacted with universal support; And

B) with described intercalator pseudonucleus thuja acid and universal support reaction; The step c) of describing in the aforesaid method is to j).

The present invention is included in simultaneously upholder bonded oligonucleotide analogs is separately removed before last sour unsettled blocking group.The optional subsequently purifying that carries out oligonucleotide analogs.

In another embodiment of the present invention, described method comprises synthetic oligonucleotide or the oligonucleotide that comprises at least one inherent intercalator pseudonucleus thuja acid, wherein synthetic can comprising the steps:

A) Nucleotide or the nucleotide analog with sour unstable protection radical protection contacts with the upholder bonded Nucleotide, oligonucleotide, nucleotide analog or the oligonucleotide analogs chain that are increasing; And

B) will be with the nucleotide analog of protecting group and described upholder bonded Nucleotide, oligonucleotide, nucleotide analog or the oligonucleotide analogs chain reaction that is increasing; And

C) product from the upholder rinses out excess reactant; And

D) randomly unreacted described upholder bonded Nucleotide is added cap; And

F) product from the upholder rinses out excess reactant; And

G) randomly unreacted described upholder bonded Nucleotide is added cap; And

H) remove the unsettled protecting group of disacidify; And

I) product from the upholder rinses out excess reactant; And j) repeating step is a)-f) to obtain the oligonucleotide analogs sequence of expectation; And

K) separate oligonucleotide analogs and remove alkali labile protecting group the alkaline medium from solid support; And

I) purifying comprises the oligonucleotide of sour unstable protection base; And

M) remove the unsettled protecting group of disacidify; And

N) obtain the oligonucleotide analogs that an intercalator is modified.

Perhaps, can before being separated, upholder bonded oligonucleotide analogs remove last sour unstable protection base.The optional purifying that carries out oligonucleotide analogs.The oligonucleotide that comprises intercalator pseudonucleus thuja acid

An object of the present invention is to provide and comprise at least one oligonucleotide or oligonucleotide analogs of pseudonucleus thuja acid as mentioned above.For example, the present invention relates to by known arbitrary method synthetic oligonucleotide of arbitrary method or person skilled in the art or oligonucleotide analogs as mentioned above.

To the high affinity of target nucleic acid can promote greatly check and analysis and in addition synthetic the nucleic acid that target nucleic acid has high affinity be can be used for many other purposes, such as gene targeting and purification of nucleic acids.The oligonucleotide or the oligonucleotide analogs that contain intercalator have shown that the homology complementary nucleic acid is had the enhanced affinity.

Therefore a purpose of the present invention provides oligonucleotide and/or the oligonucleotide analogs that comprises at least a intercalator pseudonucleus thuja acid, wherein the melting temperature(Tm) of the crossbred of forming by described oligonucleotide or oligonucleotide analogs and homology complementary DNA (DNA crossbred) be significantly higher than by the oligonucleotide of the shortage intercalator pseudonucleus thuja acid of forming with described oligonucleotide or the identical nucleotide sequence of oligonucleotide analogs or oligonucleotide analogs with and the melting temperature(Tm) of the crossbred (corresponding D NA crossbred) of described homology complementary DNA.

Preferably, the melting temperature(Tm) of DNA crossbred is from 1 to 80 ℃, more preferably at least 2 ℃, and more preferably at least 5 ℃, however more preferably high 10 ℃ than the melting temperature(Tm) of corresponding DNA crossbred at least.

The present invention also can provide oligonucleotide or the oligonucleotide analogs that comprises at least a inherent intercalator pseudonucleus thuja acid.Location intercalator internal institution allows design to go up greater flexibility.Therefore, comprise that inherent nucleic acid analog of locating intercalator pseudonucleus thuja acid can not have higher affinity than not having inherent location intercalator pseudonucleus thuja acid to the homology complementary nucleic acid.The oligonucleotide or the oligonucleotide that comprise at least one inherent intercalator pseudonucleus thuja acid also may be distinguished RNA (comprising RNA class nucleic acid analog) and DNA (comprising DNA class nucleic acid analog).Positioned internal fluorescence intercalator monomer can obtain to use in diagnostic tool in addition.

For example this oligonucleotide analogs can comprise 1, such as 2, and for example 3, such as 4, for example 5, such as from 1 to 5, such as, for example from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as surpassing 20 intercalator pseudonucleus thuja acids.

In one embodiment, oligonucleotide or oligonucleotide analogs comprise at least 2 intercalator pseudonucleus thuja acids.

Intercalator pseudonucleus thuja acid can be positioned on the site of arbitrary expectation in a given oligonucleotide or the oligonucleotide analogs.For example, an intercalator pseudonucleus thuja acid can be positioned at the end of oligonucleotide or oligonucleotide analogs, and perhaps an intercalator pseudonucleus thuja acid can be positioned at the site, inside in oligonucleotide or the oligonucleotide analogs.

When oligonucleotide or oligonucleotide analogs comprise that when surpassing 1 intercalator pseudonucleus thuja acid, intercalator pseudonucleus thuja acid can be positioned at the arbitrary position that is relative to each other.For example they can be located against each other, and perhaps they can make 1, such as 2, and for example 3, such as 4, for example 5, such as surpassing 5 sites that Nucleotide separates intercalator pseudonucleus thuja acid.In a preferred embodiment, time positioned adjacent of two intercalator pseudonucleus thuja acids in oligonucleotide or the oligonucleotide analogs, be arbitrary site that they can be positioned at oligonucleotide or oligonucleotide analogs, and have 1 Nucleotide separately described two intercalator pseudonucleus thuja acids.In another preferred embodiment, two intercalators are positioned at or are close in most each end of described or oligonucleotide analogs.

Oligonucleotide or oligonucleotide analogs can comprise the Nucleotide and/or the nucleotide analog of arbitrary kind, such as above-mentioned Nucleotide and/or nucleotide analog.For example, oligonucleotide or oligonucleotide analogs can comprise Nucleotide and/or nucleotide analog, comprise DNA, RNA, LNA, PNA, ANA and HNA.Therefore, oligonucleotide or oligonucleotide analogs can comprise the subunit of one or more following groups: PNA, homotype-DNA, b-D-pyrans altrose base-NA, b-D-glucopyranosyl-NA, b-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, be that oligonucleotide analogs can be selected from PNA, homotype-DNA, b-D-pyrans altrose base-NA, b-D-glucopyranosyl-NA, b-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-and TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA and composition thereof.

An advantage of oligonucleotide of the present invention or oligonucleotide analogs by by the oligonucleotide of at least one intercalator pseudonucleus thuja acid or oligonucleotide analogs and basically the melting temperature(Tm) of the crossbred (DNA crossbred) formed of complementary DNA be significantly higher than by complementary DNA basically with and the melting temperature(Tm) of the complementary duplex of forming of DNA.

Therefore, oligonucleotide of the present invention or oligonucleotide analogs can with have than natural exist nucleic acid more the DNA of high affinity form crossbred.Melting temperature(Tm) preferably is increased to 30 ℃ from 2, for example from 5 to 20 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, such as from 15 ℃ to 20 ℃, for example from 20 ℃ to 25 ℃, such as from 25 ℃ to 30 ℃, for example from 30 ℃ to 35 ℃, such as from 35 ℃ to 40 ℃, for example from 40 ℃ to 45 ℃, such as higher to 50 ℃ from 45 ℃.

Especially, the increase of melting temperature(Tm) may be finished owing to the embedding of intercalator, because described embedding can the stabilized DNA duplex.Therefore, preferably intercalator can the nuclear base of the intercalation of DNA between.Preferably, intercalator pseudonucleus thuja acid is as protruding the insert location or as the terminal insert location (referring to as follows) in the duplex, can embedding in some nucleic acid or nucleic acid analog.

In the present invention's one specific embodiments, comprise the oligonucleotide of at least one intercalator pseudonucleus thuja acid or oligonucleotide analogs and basically the melting temperature(Tm) of the oligonucleotide of complementary RNA (RNA crossbred) or a RNA class nucleic acid analog (RNA class crossbred) or oligonucleotide analogs be significantly higher than the melting temperature(Tm) of the duplex of the described oligonucleotide analogs that comprises described RNA of complementary basically or RNA class target and do not comprise intercalator pseudonucleus thuja acid.Great majority or all intercalator pseudonucleus thuja acids of preferably described oligonucleotide or oligonucleotide analogs are located at one end or two ends.

Therefore, oligonucleotide of the present invention and/or oligonucleotide analogs can form with RNA or RNA class nucleic acid analog or RNA class oligonucleotide analogs and have the more crossbred of high affinity than the natural nucleic acid that exists.Melting temperature(Tm) preferably is increased to 20 ℃ from 2, and for example from 5 to 15 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, such as from 15 ℃ to 20 ℃ or higher.

Described embodiment special significance is that when intercalator pseudonucleus thuja acid is positioned at described oligonucleotide or oligonucleotide analogs end described intercalator pseudonucleus thuja acid will preferably only be stablized RNA and RNA class target.Yet this does not get rid of intercalator pseudonucleus thuja acid and is positioned in the oligonucleotide or oligonucleotide analogs with the hybridization of RNA or RNA class nucleic acid analog, and therefore described intercalator pseudonucleus thuja acid is positioned at the intravital zone of hybridization of formation.Thereby can carry out this process obtain some hybridization unstable or the influence hybridization after formation molecular complex in or between 2D or 3D structure.

In another embodiment of the present invention, the oligonucleotide and/or the oligonucleotide analogs that comprise one or more intercalator pseudonucleus thuja acid of the present invention can form the triplex structure (triple strand structure) that comprises described oligonucleotide and/or oligonucleotide analogs, and described oligonucleotide and/or oligonucleotide analogs are attached to homology complementary nucleic acid or nucleic acid analog or oligonucleotide or oligonucleotide analogs by the Hoogstein base pairing.

In another preferred embodiment of the present invention, described oligonucleotide or oligonucleotide analogs can increase the melting temperature(Tm) of Hoogstein base pair described in the described triplex structure.

In another more preferred of the present invention, described oligonucleotide or oligonucleotide analogs can not rely on specific sequence constraint class and are rich in purine and are rich in the melting temperature(Tm) that mode that pyrimidine nucleic acid or nucleic acid analog duplex target sequence exist increases Hoogstein base pair described in the described triplex structure.Therefore, if described oligonucleotide or oligonucleotide analogs do not have intercalator pseudonucleus thuja acid, the described Hooogstein base pairing that Hoogstein base pair described in the described triplex structure is compared described duplex target has obviously higher melting temperature(Tm).

Therefore, oligonucleotide of the present invention and/or oligonucleotide analogs can form with homology complementary nucleic acid or nucleic acid analog or oligonucleotide or oligonucleotide analogs and have the more triplex structure of high affinity than the natural nucleic acid that exists.Preferably melting temperature(Tm) is increased to 50 ℃ from 2, such as from 2-40 ℃, such as from 2 to 30 ℃, for example from 5 to 20 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, for example from 10 ℃ to 15 ℃, such as from 15 ℃ to 20 ℃, for example from 20 ℃ to 25 ℃, such as from 25 ℃ to 30 ℃, for example from 30 ℃ to 35 ℃, such as from 35 ℃ to 40 ℃, for example from 40 ℃ to 45 ℃, such as from 45 ℃ to 50 ℃.

Especially, the increase of melting temperature(Tm) may be because the embedding of intercalator is finished, because described embedding can the stabilized DNA triple helical.Therefore, preferably intercalator can embed between the nuclear base of triplex structure.Preferably, intercalator pseudonucleus thuja acid is arranged in duplex (referring to as follows) as protruding insert, can allow the embedding of duplex in some nucleic acid or the nucleic acid analog.

The triplex form can or can be carried out chain and invaded, and it is that the 3rd chain of a kind of wherein Hoogstein base pairing invaded target duplex and replacing section or all same chain to form the method for Wo Sen-Ke Like base pair with complementary strand that described chain is invaded.This process is to carry out for several purpose.

If double-strandednucleic acid or nucleic acid analog target exist and separate described target chain is impossible, infeasible or unwanted, triplex forms and/or chain is invaded applicable to detecting for oligonucleotide and oligonucleotide of the present invention so, and described detection is to invade or the two strands in complementary district is invaded and carried out by the strand in district before not the unwinding of double-strandednucleic acid or nucleic acid analog target.

Therefore, in one embodiment of the invention, provide the oligonucleotide or the oligonucleotide analogs that comprise at least one intercalator pseudonucleus thuja acid, described intercalator pseudonucleus thuja acid can be invaded the double stranded region of nucleic acid or nucleic acid analog molecule.

In a more preferred of the present invention, the oligonucleotide or the oligonucleotide analogs that comprise at least one intercalator pseudonucleus thuja acid are provided, and described intercalator pseudonucleus thuja acid can be invaded double-strandednucleic acid or nucleic acid analog in sequence-specific mode.

Further in the embodiment, the described invader oligonucleotide of at least one intercalator pseudonucleus thuja acid and/or the oligonucleotide analogs of comprising will combine with complementary strand in the sequence-specific mode higher than the affinity of strand displacement in the present invention.

In one embodiment of the invention, the melting temperature(Tm) of the crossbred of being made up of an oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid and a homology complementary DNA (DNA crossbred) is significantly higher than the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and homology complementary RNA (RNA crossbred) or RNA class nucleic acid analog target or RNA class oligonucleotide analogs target.

Described oligonucleotide can be arbitrary above-mentioned oligonucleotide analogs.For example, oligonucleotide can be DNA oligonucleotide (analogue) or the following homotype PNA that comprises at least one intercalator pseudonucleus thuja acid, homotype-DNA, b-D-pyrans altrose base-NA, b-D-glucopyranosyl-NA, b-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA oligonucleotide or it comprise the mixture of at least one intercalator pseudonucleus thuja acid.

Therefore, described oligonucleotide or oligonucleotide analogs are obviously higher to the affinity of RNA or RNA class target than described oligonucleotide or oligonucleotide analogs to the affinity of DNA.Therefore in the mixture that comprises described oligonucleotide of limited volume or oligonucleotide analogs and homology complementary DNA and homology complementary RNA or homology complementary RNA class target, oligonucleotide or oligonucleotide analogs are preferably hybridized with described homology complementary DNA.

Preferably, the melting temperature(Tm) of DNA crossbred is than at least 2 ℃ of the melting temperature(Tm) height of homology complementary RNA or RNA class crossbred, such as at least 5 ℃, for example at least 10 ℃, such as at least 15 ℃, for example at least 20 ℃, such as at least 25 ℃, for example at least 30 ℃, such as at least 35 ℃, for example at least 40 ℃, such as from 2 to 30 ℃, for example from 5 to 20 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, for example from 10 ℃ to 15 ℃, such as from 15 ℃ to 20 ℃, for example from 20 ℃ to 25 ℃, such as from 25 ℃ to 30 ℃, for example from 30 ℃ to 35 ℃, such as from 35 ℃ to 40 ℃, for example from 40 ℃ to 45 ℃, such as from 45 ℃ to 50 ℃, for example from 50 ℃ to 55 ℃, such as from 55 ℃ to 60 ℃.

In a preferred embodiment of the present invention, comprise the secondary structure hybridization of oligonucleotide or oligonucleotide analogs and the nucleic acid or the nucleic acid analog of at least one intercalator pseudonucleus thuja acid.In a more preferred, described oligonucleotide or oligonucleotide analogs can be stablized this described secondary structure that hybridizes to.Described secondary structure can be for still being not limited to stem-ring structure, and Faraday connects, reflexed (fold-back), H-knot and protrusion.In one specific embodiment, secondary structure is one stem-ring structure of RNA, is designed to make described intercalator pseudonucleus thuja acid to hybridize to the end of one of three duplexs that three approach between described secondary structure and described oligonucleotide or oligonucleotide analogs are connected to form comprising the oligonucleotide of at least one intercalator pseudonucleus thuja acid or oligonucleotide analogs.

The position of intercalator pseudonucleus thuja acid.

In the present invention's one preferred embodiment, oligonucleotide or oligonucleotide analogs design in the mode that it can hybridize to homology complementary nucleic acid or nucleic acid analog (target nucleic acid).Preferably, oligonucleotide or oligonucleotide analogs can be complementary to target nucleic acid basically.More preferably, when locating an intercalator pseudonucleus thuja acid at least with convenient oligonucleotide analogs and target nucleic acid hybridization, the position of intercalator pseudonucleus thuja acid is as protruding insert, that is, the contiguous Nucleotide in downstream of contiguous Nucleotide in the upstream of intercalator pseudonucleus thuja acid and intercalator pseudonucleus thuja acid and the adjacent nucleotide hybridization in the target nucleic acid.

In another preferred embodiment, the site of intercalator pseudonucleus thuja acid is against an end or the two ends of the duplex that forms between the oligonucleotide analogs that comprises described intercalator pseudonucleus thuja acid and target nucleotide or the nucleotide analog, and for example described intercalator pseudonucleus thuja acid can be used as Dan Geer and piles up end altogether.

More preferably, satisfy when oligonucleotide analogs and target nucleic acid hybridization the position of all intercalator pseudonucleus thuja acids of oligonucleotide or oligonucleotide analogs, and all intercalator pseudonucleus thuja acids are as protruding insert and/or piling up end altogether as Dan Geer.

In one embodiment, the present invention relates to following oligonucleotide:

N ₁-(P) _q-N ₂，

N ₁-(P-N ₃) _q-N ₂，

(P) _q-N ₂，NL-(P)Q，

(P) _q-N ₂-(P) _r，

N ₁-(P) _q-N ₂，

N ₁-(P-N ₃) _q-N ₂-(P-N ₃) _rN ₄，

N wherein ₁, N ₂, N ₃, N ₄The sequence of representing the Nucleotide and/or the nucleotide analog of at least one Nucleotide respectively,

P represents intercalator pseudonucleus thuja acid, and

Q and r are selected from the integer of 1-10 respectively.

Oligonucleotide oligonucleotide and/or similar sequence are right

The present invention also relates to comprise that the oligonucleotide or the oligonucleotide analogs of first sequence and second sequence are right, wherein first sequence is oligonucleotide and/or the oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid, second sequence can with described first sequence hybridization.In one embodiment, described second sequence does not comprise arbitrary intercalator pseudonucleus thuja acid.

Therefore, it is right to the present invention relates to corresponding oligonucleotide or oligonucleotide analogs, wherein a right oligonucleotide analogs of oligonucleotide or oligonucleotide analogs is expressed as first sequence, another right oligonucleotide analogs of oligonucleotide or oligonucleotide analogs is expressed as second sequence, and wherein said oligonucleotide or oligonucleotide analogs are to comprising at least one intercalator pseudonucleus thuja acid.

Preferably, oligonucleotide or oligonucleotide analogs sequence (being called first sequence and second sequence) to comprise can with first sequence of second sequence hybridization.Preferably first sequence and second sequence all comprise at least one intercalator pseudonucleus thuja acid in addition.

In one embodiment of the invention, first sequence is complementary to second sequence basically.In addition, in described embodiment, preferably first sequence has substantially the same length with second sequence.

Preferably, the melting temperature(Tm) of the crossbred between first sequence and second sequence significantly is lower than first sequence of above-mentioned selection and the melting temperature(Tm) of the crossbred between corresponding nucleic acids or the nucleic acid analog.Especially, preferably the melting temperature(Tm) of the crossbred of first sequence and second sequence significantly is lower than the melting temperature(Tm) of the crossbred between first sequence and the corresponding DNA.

Especially, if second sequence is complementary to first sequence and first sequence and second sequence length equates, preferably the melting temperature(Tm) of the crossbred of first sequence and second sequence significantly is lower than the melting temperature(Tm) of the crossbred between second sequence and corresponding nucleic acids or the nucleic acid analog as mentioned above.Especially, preferably the melting temperature(Tm) of the crossbred of first sequence and second sequence significantly is lower than second sequence and the melting temperature(Tm) of the crossbred between the complementary DNA basically.

Yet when second sequence only is complementary to a part of first sequence, between first and second sequences melting temperature(Tm) of crossbred might than second sequence and basically between the complementary DNA melting temperature(Tm) of crossbred higher, equate or lower.

When the melting temperature(Tm) of the crossbred of first sequence and second sequence is obviously lower than the melting temperature(Tm) of the crossbred between first sequence and the corresponding D NA, favourable effect will be produced, promptly comprise in first sequence, second sequence and the mixture corresponding to the DNA of first sequence, first sequence will be preferably with corresponding D NA but not with second sequence hybridization.

Similarly, if second be complementary to first sequence and first sequence and second sequence length equates, in comprising first sequence, the mixture of second sequence corresponding to the DNA of second sequence, second sequence will be preferably with corresponding D NA but not first sequence hybridization.Yet, comprise first sequence and second sequence and also do not comprise the nucleic acid of corresponding intercalator pseudonucleus thuja acid or the mixture of nucleic acid analog in, first sequence will with second sequence hybridization.If second sequence only is complementary to a part of first sequence, between first and second sequences melting temperature(Tm) of crossbred might than second sequence and basically between the complementary DNA melting temperature(Tm) of crossbred higher, equate or lower.

Therefore, in the mixture that comprises first sequence and second sequence, if first sequence and second sequence hybridization, this just shows that the corresponding target nucleic acid of a limited volume is effectively true.

Vice versa, and in the mixture that comprises first sequence and second sequence, if first sequence and second sequence are not hybridized, this just shows that mixture also comprises the fact of respective target nucleic acid.

Preferably, do not comprise that the corresponding nucleic acid of described intercalator pseudonucleus thuja acid or nucleic acid analog are DNA.

Melting temperature(Tm) depends on many features, for example melting temperature(Tm) depends on the amount of intercalator pseudonucleus thuja acid, the kind of intercalator pseudonucleus thuja acid, the length of first sequence and/or second sequence, the nuclear base composition, these intercalator pseudonucleus thuja acids are the position of intercalator pseudonucleus thuja acid in the position of oligonucleotide or oligonucleotide analogs sequence centering and each other.

Preferably, selecting above feature combines with the specificity of respective target nucleic acid so that guarantee.

Therefore, first nucleotide sequence preferably includes between 5 and 10, between 10 and 15, and for example between 15 and 20, between 20 and 30, for example Nucleotide between 30 and 50 and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.More preferably, first nucleotide sequence comprises between 5 and 10, between 10 and 15, and for example between 15 and 20, between 20 and 30, for example Nucleotide between 30 and 50 and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.

In addition, second nucleotide sequence preferably includes between 5 and 10, between 10 and 15, and for example between 15 and 20, between 20 and 30, for example Nucleotide between 30 and 50 and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.More preferably, second nucleotide sequence comprises between 5 and 10, between 10 and 15, and for example between 15 and 20, between 20 and 30, for example Nucleotide between 30 and 50 and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.

In a preferred embodiment, first nucleotide sequence and second nucleotide sequence comprise Nucleotide and/or the nucleotide analog and/or the intercalator pseudonucleus thuja acid of same amount.

Each comprises Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid between 5 and 100 respectively to described oligonucleotide analogs in addition, preferably, oligonucleotide or oligonucleotide analogs can comprise Nucleotide and/or nucleotide analog and/or the intercalator pseudonucleus thuja acid between 10 and 75, more preferably, oligonucleotide or oligonucleotide analogs can be at the Nucleotide between 15 and 50 and/or nucleotide analog and/or intercalator pseudonucleus thuja acids.

First sequence should comprise at least one intercalator pseudonucleus thuja acid, for example 2, and such as 3, for example 4, such as 5, for example 6, such as from 6 to 10, for example from 10 to 15, such as from 15 to 20 intercalator pseudonucleus thuja acids.

Second sequence can or can not comprise intercalator pseudonucleus thuja acid.In a preferred embodiment of the invention, especially when second sequence and the first sequence complementation and equal in length, preferably second sequence comprises at least one intercalator pseudonucleus thuja acid, such as 1, for example 2, such as 3, for example 4, such as 5, for example 6, such as from 6 to 10, for example from 10 to 15, such as from 15 to 20 intercalator pseudonucleus thuja acids.

Intercalator pseudonucleus thuja acid can be positioned at the site of the first and/or second arbitrary expectation of sequence.For example intercalator pseudonucleus thuja acid may be positioned at the end of first and/or second sequence or the site, inside that intercalator pseudonucleus thuja acid may be positioned at first and/or second sequence.

In addition,, first sequence surpasses one intercalator pseudonucleus thuja acid, the location that described pseudonucleus thuja acid can arbitrary desirable mode be relative to each other if comprising.For example, they may make 1, and for example 2, such as 3, for example 4, such as 5, for example from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as surpassing 20 Nucleotide the mode that intercalator pseudonucleus thuja acid separates is located.

Similarly,, first sequence surpasses one intercalator pseudonucleus thuja acid, the location that described pseudonucleus thuja acid can arbitrary desirable mode be relative to each other if comprising.For example, they may make 1, and for example 2, such as 3, for example 4, such as 5, for example from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as surpassing 20 Nucleotide the mode that intercalator pseudonucleus thuja acid separates is located.

In a preferred embodiment, at least 2 intercalator pseudonucleus thuja acids are relative to each other and are positioned at first sequence and/or second sequence so that obtain selectivity and the affinity higher to corresponding target nucleic acid.Therefore, preferably the localized position of intercalator pseudonucleus thuja acid satisfy oligonucleotide analogs preferably with corresponding target sequence but not with arbitrary other nucleic acid array hybridizing of the simple point mutation that comprises described respective target nucleic acid.For example, in one embodiment of the invention, intercalator pseudonucleus thuja acid can be close in proper order.

First and/or second sequence can comprise respectively and surpass one intercalator pseudonucleus thuja acid, wherein said intercalator pseudonucleus thuja acid can similar or described intercalator pseudonucleus thuja acid can be different.

If particularly second sequence is complementary to first sequence and equal in length therewith, the intercalator pseudonucleus thuja acid in first sequence how with second sequence in intercalator pseudonucleus thuja acid located in connection just very important.Preferably, when first sequence and second sequence hybridization, the interior localized mode of at least one intercalator pseudonucleus thuja acid of first sequence satisfies it and is positioned at the opposite site that replaces with the second sequence kernel thuja acid that can not form the Wo Senkelike hydrogen bond.In addition, preferably the localized mode of at least one intercalator pseudonucleus thuja acid of second sequence satisfies when first sequence and second sequence hybridization, and it is positioned at first consecutive nucleotides that can not form the Wo Senkelike hydrogen bond and replaces opposite site.

It can for example be the Nucleotide of a shortage nuclear base or the Nucleotide that comprises a nuclear base that described Nucleotide replaces, and described nuclear base is modified in the mode that it no longer forms the Wo Senkelike hydrogen bond.Yet Nucleotide is substituted by another intercalator pseudonucleus thuja acid of describing here in a preferred embodiment.

After hybridization they each other closely near the time first and second sequences Nucleotide, Nucleotide replacement, nucleotide analog and intercalator pseudonucleus thuja acid it is said mutually positioning opposite.Preferably, when they were directly opposite one another, Nucleotide, Nucleotide replacement, nucleotide analog and intercalator pseudonucleus thuja acid it is said that the location is opposite each other.Yet, when they be positioned at around the directly opposite intercalator pseudonucleus thuja acid/Nucleotide replacements/nucleotide analog/intercalator pseudonucleus thuja acid of Nucleotide/Nucleotide replacement/nucleotide analog/described Nucleotide than the zonule in the time, Nucleotide, Nucleotide replacement, nucleotide analog and intercalator pseudonucleus thuja acid it is said that also the location is on the contrary.An Examples set of phase antidirection finding intercalator pseudonucleus thuja acid has shown that wherein the oligonucleotide analogs that comprises phase antidirection finding intercalator pseudonucleus thuja acid is right in Figure 14.

Therefore, preferably the localized mode of at least one intercalator pseudonucleus thuja acid of first sequence satisfies when first sequence hybridization during to second sequence, and it is positioned at least one opposite site of intercalator pseudonucleus thuja acid of second sequence.

More preferably, the localized mode of each intercalator pseudonucleus thuja acid of second sequence satisfies when first sequence hybridization during to second sequence, and they are positioned at the opposite site of intercalator pseudonucleus thuja acid of first sequence.

Another object of the present invention provides a system, and wherein first sequence is connected with second sequence.

First sequence and second sequence can be each other directly or be connected indirectly, for example they covalently bound each other or they to pass through the 3rd sequence covalently bound, perhaps they can only be connected to each other when they are for example hybridized by hydrogen bond.

Therefore, two sequences can be included in the nucleic acid analog.Perhaps, first nucleotide sequence may be included in first nucleic acid or the nucleic acid analog and second nucleotide sequence may be included in second nucleic acid or the nucleic acid analog.

When first sequence and second sequence were included in the oligonucleotide analogs, so described oligonucleotide analogs was preferably with the same with second sequence length altogether with first sequence at least; Yet oligonucleotide analogs may be longer altogether than first sequence and second sequence, and therefore oligonucleotide analogs can comprise other other parts except that first sequence and second sequence.

For example, oligonucleotide analogs preferably includes between 5 and 100, between 5 and 10, between 10 and 15, for example between 15 and 20, between 20 and 30, for example between 30 and 40, between 40 and 50, for example between 50 and 60, between 60 and 80, for example Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid between 60 and 100.More preferably oligonucleotide analogs comprises the Nucleotide of 15 to 50 scopes.

In one embodiment, an oligonucleotide analogs can comprise first sequence and corresponding second sequence, and wherein said first sequence and described second sequence comprise that by one the 3rd sequence of P Nucleotide and/or nucleotide analog separates.

P can be arbitrary desirable integer, and for example p may be the integer between 1 and 5, for example 5 and 10, and between 10 and 15, for example between 15 and 20, between 20 and 30, the integer between 30 and 50 for example.

Oligonucleotide analogs of the present invention can comprise the intercalator pseudonucleus thuja acid of arbitrary desirable quantity.For example oligonucleotide analogs can comprise between 2 and 5, between 5 and 10, and between 10 and 15, the intercalator pseudonucleus thuja acid of quantity between 15 and 20 for example.

Intercalator pseudonucleus thuja acid can be dispersed in arbitrary site of first, second and/or the 3rd sequence of oligonucleotide analogs.

The oligonucleotide that comprises fluorophor

In one embodiment, oligonucleotide analogs of the present invention carries out mark with detectable mark.The intercalator pseudonucleus thuja acid that for example is included in the oligonucleotide analogs usually can have fluorescent characteristic, especially comprise many can fluorescigenic intercalator.

In some embodiments of the present invention, the oligonucleotide that comprises at least one intercalator pseudonucleus thuja acid can comprise the fluorophor that at least one is other.For example fluorophor can be selected from and include but not limited to fluorescein, FITC, rhodamine, lissamine rhodamine, rhodamine 123, acridine orange, tonka bean camphor, CY-2, CY-3, CY3.5, CY-5, CY5.5, ethidium bromide, FAM, GFP, YFP, BFP, YO-YO, HEX, JOE, the Nano orange, Nile red, OliGreen, the Oregon is green, Pico is green, Propidium iodine, radiation is red, ribose is green, ROX, the R-phycoerythrin, the SYBR Huang, the green I of SYBR, the green II of SYBR, the SYPRO orange, SYPRO is red, SYPRO is dark red, TAMRA, texas Red and XRITC.

In a preferred embodiment, be labeled as the mixture of at least two intercalator pseudonucleus thuja acids of the present invention, can form excimer, exciplex, FRET or charge-transfer complex in a part.

In addition, comprise also in the scope of the present invention that oligonucleotide analogs of the present invention can comprise at least one quencher molecule.

Quencher molecule of the present invention is the molecule of arbitrary fluorescence that can its adjacent specific fluorophor of quencher.Quencher can be by absorbing energy and bringing into play function with the form dissipation energy of heat or radiation damping from fluorophor.Therefore, the signal from fluorophor may reduce or lack.Therefore, if fluorophor and suitable quencher molecule location closer to each other, the fluorescence of fluorophor may quencher.

The example of quencher molecule is including but not limited to DABCYL, DABSYL TAMRA, methyl red, black HOLE-1, black Hole-2, ElleQuencher and QSY-7.Yet quencher molecule should be selected according to fluorophor usually.

A preferred embodiment of the present invention provides the oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid and its and comprises also that in addition a fluorophor and can quench fluorescence roll into a ball fluorescigenic quencher molecule.

Preferred fluorophor quencher molecule of the present invention to comprising but not office in:

Fluorophor Quencher

FAM???????????????????TAMRA

TET???????????????????TAMRA

Rhodamine TAMRA

Tonka bean camphor DABCYL

EDANS?????????????????DABCYL

Fluorescein DABCYL

Fluorescent yellow DABCYL

Eosin DABCYL

TAMRA?????????????????DABCYL

Fluorophor and quencher molecule can be positioned at arbitrary position of nucleic acid respectively.Yet in a preferred embodiment, at least one fluorophor is connected free end, and more preferably all fluorophors that are not included in the intercalator pseudonucleus thuja acid are connected free end.Fluorophor can be positioned at the free end of 5 ' end or be positioned at 3 ' end or two ends.Equally preferably, at least one quencher molecule is connected free end, and more preferably all quencher molecule that are not included in the intercalator pseudonucleus thuja acid are connected free end.Quencher molecule can be used as 5 ' terminal free end and locatees or be positioned at 3 ' end or two ends.

A preferred embodiment of the present invention provides a pair of probe that the present invention includes first sequence and corresponding second sequence.Preferably first sequence comprise a fluorophor and the second sequence preference ground comprise one can the described fluorophor of quencher quencher molecule.Therefore, do not have only when first nucleic acid hybridizes to second nucleic acid and just can detect.

Perhaps, each sequence (first sequence and second sequence) can comprise a fluorophor and a quencher, and wherein the fluorophor of first sequence can be by being included in the quencher in first sequence and/or being included in quencher quencher in second sequence; The fluorophor of second sequence that vice versa can be by being included in article one intrachain quencher and/or being included in the quencher of second intrachain quencher.

Especially, when second sequence was included in second nucleic acid in first sequence is included in first nucleic acid, first nucleic acid can comprise that at least one fluorophor and/or second nucleic acid can comprise at least one fluorophor.

Can use and surpass one fluorophor in order to obtain stronger signal, for example first sequence can comprise that two fluorophors and second nucleic acid can comprise two quencher molecule.Preferably, when first sequence and second sequence are hybridized mutually, quencher molecule and fluorophor so that each quencher group can a fluorophor fluorescence of quencher mode position.

In a more preferred, oligonucleotide analogs comprises first sequence and corresponding second sequence of being separated by the 3rd sequence (a hair clip probe), wherein one of sequence comprises that at least one intercalator pseudonucleus thuja acid and oligonucleotide analogs have an other fluorophor unsettled end terminal as 5 ' or 3 ' end and locate at least, and other quencher molecule is positioned at the end opposite with described fluorophor as unsettled end.

For the length of intercalator pseudonucleus thuja acid, complementary degree number and position are some parameters that can change so that obtain the melting temperature(Tm) expected between first and second sequences.

Therefore unless a preferred embodiment of the present invention provides one run into the hair clip probe that complete complementary target can oneself's hybridization under hybridization conditions.

When mark was the mixture of at least two intercalator pseudonucleus thuja acids that can form intramolecularly excimer, exciplex, FRET or charge-transfer complex (referring to following), preferably at least two intercalator pseudonucleus thuja acids were separated by at least one Nucleotide or nucleotide analog.In such embodiment, can be by the Nucleotide of at least one Nucleotide in the mixture in the zone of the both sides of arbitrary intercalator pseudonucleus thuja acid and the quencher of the hybridization picked up signal of complementary sequence.

Therefore a preferred embodiment of the present invention provides an oligonucleotide analogs that comprises at least two intercalator pseudonucleus thuja acids, wherein hybridizing to target nucleic acid or since target nucleic acid amplification back spectral response curve change.In a preferred embodiment, spectral signal is not lower when having or have only a small amount of target nucleic acid, and when have a large amount of target nucleic acids when existing spectral signal higher.When during the target sequence amplified reaction, for example when using during the pcr amplification, the preferably increase of the corresponding described target nucleic acid sequence of spectral signal and increasing.

Therefore, preferably the invention provides and comprise by the 3rd sequence (a hair clip probe) first sequence of separating and the oligonucleotide analogs of complementary second sequence, wherein the second or the 3rd sequence comprise at least one intercalator pseudonucleus thuja acid and wherein first sequence comprise the other mixture of intercalator pseudonucleus thuja acid of the present invention, wherein when described first sequence hybridization during to second sequence spectral signal lower and when they are not hybridized spectral signal higher.

Fluorescent energy resonance transfer (FRET) is the interaction that relies on distance between the excited electronic state of two dye molecules, wherein transfers to an acceptor molecule without exciting from a donor molecule of photo emissions.FRET depends on intermolecular isolating reverse 6th energy, makes it can be according to using with the distance of biopolymer coupling.Preferably, for FRET takes place, donor and acceptor must be closely near (generally between 10 to 100 ).In addition, the absorption spectrum of acceptor must be consistent with the fluorescence emission spectrum of donor.Further preferably donor and acceptor migration dipole orientation must almost parallel.

Comprise also in the scope of the present invention that first sequence can comprise the donor of FRET and the acceptor that second sequence can comprise FRET.Satisfy when first sequence hybridization position of preferably described donor and described acceptor FRET can take place during to second sequence.It is right that fluorophor can for example be used to detect the oligonucleotide analogs sequence.

FRET donor and acceptor are right, for example comprise:

Donor Acceptor

The fluorescein tetramethylrhodamin

The IAEDANS fluorescein

EDANS???????????DABCYL

The fluorescein fluorescein

BODIPY?FL???????BODIPY?FL

Fluorescein QSY7 dyestuff

In a preferred embodiment, at least two intercalator pseudonucleus thuja acid time neighbour's atom location, promptly Nucleotide is with intercalator pseudonucleus thuja acid separately.

Therefore an object of the present invention is to provide an oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid, can be used for detecting the existence of target nucleic acid sequence comprising the spectral response curve in described oligonucleotide analogs.In a preferred embodiment, during the amplified reaction of target nucleic acid sequence, can detect in real time, perhaps adopt owing to hybridize to the advantage of the spectral response curve that the target nucleic acid sequence of described oligonucleotide analogs changes or by utilizing the activity of the circumscribed or restriction endonuclease of 5 ' of archaeal dna polymerase-3 ' nucleic acid, described archaeal dna polymerase can increase the spectral signal from the arbitrary probe of the present invention.

In addition or other fluoroscopic examination can carry out after the so-called end-point detection at amplification procedure.

Detect the method for hybridization

One embodiment of this invention relates to the detection target nucleic acid and comprises the method for hybridizing between first sequence of at least one intercalator pseudonucleus thuja acid, and described intercalator pseudonucleus thuja acid has the structure of following general formula:

X-Y-Q

Wherein

X is the backbone monomers unit that can be embedded into above-mentioned nucleic acid main chain; And

Q is the intercalator that comprises at least one flat basically conjugate system, and described conjugate system can be piled up altogether with above-mentioned nucleic acid or nucleic acid analog; And

Y one connects the described backbone monomers unit and the shank of described intercalator as mentioned above;

Described method comprises the steps:

A) provide target nucleic acid and complementary target nucleic acid randomly; With

B) provide at least a oligonucleotide analogs that comprises described first sequence, wherein first sequence can be hybridized with described target nucleic acid; With

C) with target nucleic acid and oligonucleotide or oligonucleotide analogs incubation together under hybridization conditions; With

D) detect hybridization.

In the present invention's one preferred embodiment, first sequence comprises at least two intercalator pseudonucleus thuja acids, and each comprises that one can form the intercalator of excimer, exciplex or charge-transfer complex.

Preferably do not hybridize and described intercalator can not form intramolecularly excimer, intramolecularly exciplex, intramolecularly FRET mixture or charge-transfer complex the time when first sequence, when described first sequence hybridization to a corresponding nucleic acids or nucleic acid analog, more preferably when at least one separates the Nucleotide of intercalator pseudonucleus thuja acid and complementary nucleotide hybridization, the intercalator of at least two intercalator pseudonucleus thuja acids can form in a part exciplex, intramolecularly FRET mixture or a charge-transfer complex in excimer, a part.Preferably have only a Nucleotide to separate described intercalator pseudonucleus thuja acid.

Therefore, the hybridization of first sequence and arbitrary corresponding sequence can be determined by determining that excimer fluorescence, exciplex fluorescence, intramolecularly FRET mixture fluorescence or charge transfer absorb in described embodiment.Described oligonucleotide analogs can be preferably designed so that strong excimer fluorescence, exciplex fluorescence, FRET fluorescence or the charge transfer described oligonucleotide analogs that absorbs that performance is not hybridized or vice versa can be designed to strong excimer, exciplex or charge transfer and absorb and be used for performance hybridization.

Excimer is the dipolymer of compound, and the relevant association of its excited state with electronics and its are dissociated in ground state.When isolated compound was excited, it may be lost it and excites or it can combine with another similar compound (not exciting), forms excimer thus.Be different from the monomer fluorescence emitted fluorescence a wavelength excimer emission.When excimer is lost it and is excited, in conjunction with no longer suitable and two kinds all will dissociate.Exciplex is an excimer bi-polymer analogy, and wherein two compounds are different.

The intramolecularly excimer is included in an intramolecular part by two and forms, for example the aromatic group more than two in the same molecular.Be included in intramolecular two parts, for example form similar intramolecularly exciplex by 2 different many aromatic groups.

Charge-transfer complex wherein has the weak coordination that participates in charge transfer between two molecules.An example is phenol and benzoquinones, and wherein phenol and quinone molecule are not to close but by the transfer bonded of electric charge between the aromatic nucleus system of compound by formal chemical bonded refractory.

In another embodiment of the present invention, in the step b) of above-mentioned detection hybridizing method, provide aforesaid a pair of oligonucleotide analogs sequence as an alternative.

Preferably, target nucleic acid comprise one can with the sequence of first sequence hybridization and complementary target nucleic acid comprise one can with the sequence of right second nucleotide sequence hybridization of oligonucleotide analogs as mentioned above.

Especially, preferably use a pair of oligonucleotide or the oligonucleotide analogs that comprises aforesaid fluorophor and/or quencher molecule.For example first sequence can comprise that aforesaid fluorophor and/or quencher molecule and/or second sequence can comprise aforesaid fluorophor and/or quencher molecule.

Therefore can determine by the spectral response curve of determining first sequence and/or the spectral response curve of determining second sequence.

Especially spectral response curve can be a fluorescent characteristic, and preferably described fluorescent characteristic is the fluorescence of non-intercalator pseudonucleus thuja acid.

In a preferred embodiment of the present invention, when first sequence and second sequence are hybridized each other (referring to above-mentioned), the fluorophor of first sequence will be near the quencher group of second sequence.Therefore, if first sequence and second sequence hybridization will not have detection signal; Yet if first sequence and corresponding nucleic acids hybridization, promptly target nucleic acid will have detection signal.

In described embodiment, therefore a spectral signal that is higher than predetermined threshold can show hybridization.

Perhaps, can determine hybridization by determining melting temperature(Tm).Can determine like this because the melting temperature(Tm) of the crossbred between first sequence and second sequence than comprising that first sequence and one do not comprise that the melting temperature(Tm) of crossbred of the corresponding nucleic of described intercalator pseudonucleus thuja acid or nucleic acid analog is low.

Therefore, low melting temperature(Tm) shows the hybridization between first and second sequences, and higher melting temperature(Tm) shows the hybridization between first sequence and corresponding nucleic acids and/or second sequence and the corresponding nucleic acids.

Therefore the melting temperature(Tm) that is higher than predetermined threshold can show by hybridization.

The method that detects hybridization can be used for different purposes.For example, described method can be used for the quantitative analysis polymerase chain reaction.

Perhaps, described method can be used for detecting the analysis that depends on specific hybrid, Southern blotting for example, Northern blotting, the hybridization in the in situ hybridization of FISH or other kind.

Perhaps, can use probe, need for example by cleaning the probe of removing non-specific binding with non-quencher mark.

The method of nucleotide sequence during the real-time detection amplified reaction

The present invention also relates to the method for during amplified reaction real time nucleic acid detection or nucleic acid analog sequence, described method comprises the steps:

A) provide at least one to comprise the template that one or more needs amplifying nucleic acid sequence; With

B) provide at least one aforesaid oligonucleotide analogs sequence, wherein said oligonucleotide analogs can with the nucleic acid array hybridizing of needs amplifications; With

C) provide at least one cover can with the primer of the template nucleic acid complementary nucleic acid hybridization of needs amplifications; With

D) described template nucleic acid and described oligonucleotide analogs and described primer series are incubated under hybridization conditions; With

E) Ren Xuan detection; With

F) 5 ' prolong described primer to 3 ' direction in the mode that relies on template; With

G) Ren Xuan detection.

This method can comprise the steps: in addition

A) under the condition that can not hybridize, cultivate the mixture that comprises nucleic acid and nucleic acid analog; With

B) repeating step d), e), f), g) with optional step h).

Each step may be carried out several times.Especially, step d), e), f), g) and h) can repeat at least once, for example between 2 and 5 times, such as between 5 and 10 times, such as between 10 and 15 times, for example between 15 and 20 times, such as between 20 and 30 times, for example between 30 and 50 times.

Primer can be arbitrary nucleic acid or the nucleic acid analog sequence between 5 and 100 base pairs are long preferably.

The amplification of specific nucleic acid sequence for example can be polymerase chain reaction (PCR).

Usually, the PCR temperature cycle is included at least two kinds of cultivations of differing temps.One during these are cultivated is to be used for primer hybridization and catalytic primer extension reaction.Another cultivation is to be used for sex change, is about to double-stranded extension products and is separated into the single-stranded template that is used for hybridizing next time and prolonging the cultivation interval.

The details of polymerase chain reaction, temperature cycle that PCR is required and reaction conditions and all ingredients and the enzyme that react required for example are described in United States Patent (USP) 4,683,202,4,683,195, EPO disclosed 258,017 and 4, in 889,818 (the Taq polysaccharase patents), be incorporated herein by reference herein.

Yet more continually, PCR comprises initial denaturing step, its with chain of double chain target acid sample separately, succeeded by the repetition of following steps: 1. annealing steps makes the amplimer specificity be annealed to the opposite strand and the flank target sequence site of target; 2. one extends step and in the mode that can detect template primer 5 ' is extended to 3 ', thus form target complementary copy and; 3. a denaturing step makes copy separate with target.Each above-mentioned steps can be carried out in differing temps, wherein can utilize thermocirculator to finish the change of temperature.Produce duplicating of index stage by the simple temperature cycle repeating step of sample 1-3, usually at 20-40 the circulation millions of copies of generation or the target duplex (Innis etc. of multiple copied more, the PCR scheme: methods and applications instruct, (1990) Academic Press, Saiki etc., PCR Protocols:A Guide to Methods andApplications Science, (1988) 239:487).

The purpose of polymerase chain reaction is to produce identical or a large amount of DNA of a small amount of " seed " DNA that provides at first is provided.Described reaction comprises that copy DNA chain utilizes copy to produce other copy then in circulation subsequently.Under ideal conditions, therefore each circulation DNA amount that will double to provide causes a large amount of copies of " target " or " seed " DNA chain geometricprogression that is present in the reaction mixture.Yet PCR also can be used for introducing sudden change and increasing them.

An example of typical PCR program is as described below: program originates in 94 ℃ sample temperature and keeps making in 30 seconds the reaction mixture sex change.Then, the temperature of reaction mixture is reduced to the temperature from 35 ℃ to 65 ℃ of scopes and remains in 15 seconds to 2 minutes the scope and makes primer hybridization.Secondly, the temperature of reaction mixture is elevated to the temperature from 50 ℃ to 72 ℃ of scopes, and wherein it remains in 30 seconds to 5 minutes the scope to promote the synthetic of extension products.This has finished one-period.The chain that is used for the extension products of previous circulation (sex change) formation by the next PCR circulation of temperature to 94 ℃ beginning that improves reaction mixture separates then.Usually, circulation repeats 20 to 50 times.

Usually, need as far as possible promptly in circulation, to change sample temperature for several reasons to next temperature.At first, chemical reaction has the optimum temperuture in its middle each stage.Therefore, consuming the less time in non-optimum temperuture means and has realized the better chemical result.Another reason is need keep the minimum time of reaction mixture in each culture temperature after each described culture temperature reaches.These minimum incubation times have determined to finish " substrate " or the minimum time that circulation is required.Arbitrary time of changing between the sample cultivation temperature is the time that increases this minimum cycle time.Because cycle number is quite big, this other time has unnecessarily prolonged the total time that need finish amplification.

In addition, more than amplification can comprise the step of determining a pair of nucleotide sequence fluorescent characteristic.Determine that spectral response curve can for example comprise definite one or more and be selected from the group that monomer fluorescence, excimer fluorescence, exciplex fluorescence, FRET and charge transfer absorb.Especially, spectral response curve can comprise monomer fluorescence, excimer fluorescence, exciplex fluorescence, FRET fluorescence or the charge transfer absorption of the intercalator of intercalator pseudonucleus thuja acid.

Perhaps, it is definite in conjunction with the right FRET of the donor/acceptor of first and/or second sequence to determine that spectral response curve can comprise.

An advantage of the inventive method is to determine spectral response curve when carrying out amplified reaction.Therefore the information of spectral response curve can be used for determining for example round-robin number, the length in per step, and the temperature of each step between the reaction period, and therefore reaction can be adjusted to concrete needs.

In a preferred embodiment, the oligonucleotide analogs that comprises mark hybridizes to template nucleic acid during the extension step of described method.Therefore can utilize endonuclease and/or the 5 '-3 ' exonuclease activity of archaeal dna polymerase rupture in the described oligonucleotide analogs main chain key and therefore they are separated.Especially, the least part that preferably separates comprises the mark of described oligonucleotide analogs, and this has increased the detection signal from described mark.

In another preferred embodiment, the oligonucleotide analogs that comprises a mark only hybridizes to template nucleic acid being lower than under the temperature of elongating temperature.In this temperature, if preferably described oligonucleotide analogs does not hybridize to target nucleic acid sequence, in described oligonucleotide analogs from the signal of certification mark by quencher.Most preferably, measure detection signal and be used to extend under the temperature of step at described low temperature and do not hybridize described oligonucleotide analogs.

The method that regulatory gene is transcribed

The present invention relates to the method that one or more specific gene of adjusting is transcribed in addition, and described method comprises the steps:

E) provide a re-reading system; And

F) provide the oligonucleotide and/or the oligonucleotide analogs of first sequence of an at least a intercalator pseudonucleus thuja acid that comprises at least a following formula,

X-Y-Q

Wherein

X is the backbone monomers unit that can embed the phosphoric acid ester main chain of nucleic acid; And Q one comprises the intercalator of the conjugate system that at least one is generally planar, and the described conjugate system of nuclear base can be piled up altogether with the nuclear base of nucleic acid; And

Y is the shank of a connection described backbone monomers unit and described intercalator; And wherein said first sequence can be regulated the complementary strand hybridization that sequence or described gene and/or its are regulated sequence with described gene and/or its; With

G) described first sequence is embedded re-reading system; With

H) with first sequence and described one or more gene and/or its complementary strand hybridization of regulating sequence or gene and/or regulating sequence; And

Therefore regulate described gene transcription.

Described oligonucleotide or oligonucleotide analogs can comprise that one or more is selected from the subunit of DNA, RNA, LNA, PNA, ANA, 2 '-O-methyl RNA, MNA and HNA.

Gene transcription is preferably regulated because oligonucleotide can with gene, the sequence hybridization of complementary strand and/or regulatory gene.The adjusting of transcribing in one embodiment of the invention, is based on the inverted defined gene strategy.In the inverted defined gene strategy, hybridization can for example cause the spatial interference of the mechanism of transcribing and therefore cause the blocking-up that stops or transcribing of transcribing in one embodiment of the invention.

Usually the inverted defined gene strategy comprises that chain invades, and the meaning refers to that oligonucleotide or oligonucleotide analogs can invade double chain DNA molecule, and with a chain hybridization.Therefore, preferably oligonucleotide or oligonucleotide analogs have the affinity higher to DNA than DNA to DNA.

In the present invention's one preferred embodiment, the inverted defined gene strategy comprises double-stranded the intrusion.For example, strategy comprises and utilizes a pair of oligonucleotide or oligonucleotide analogs, and wherein each oligonucleotide and/or oligonucleotide analogs can hybridize to gene and/or its and regulate sequence or gene and/or its complementary strand of regulating sequence and have and regulate sequence than gene and/or its its complementary strand is had higher affinity and oligonucleotide or oligonucleotide analogs to having higher affinity each other.

Double-stranded principle of invading is described among Figure 15.

In a preferred embodiment of the present invention, the step b) of regulating dubbing method comprises provides a pair of aforesaid Nucleotide and/or nucleotide analog sequence, wherein first sequence can with gene and/or its regulate sequence hybridization and second sequence can be complementary to gene/or its regulate the nucleic acid array hybridizing of sequence.

In this embodiment, described Nucleotide and/or nucleotide analog sequence are to embedding the re-reading system and first sequence and gene or its adjusting sequence hybridization and second sequence and the hybridization of another chain.

Utilize Nucleotide of the present invention and/or nucleotide analog sequence to have several advantages to regulating genetic expression.For example, many organisms or cell type comprise the defense mechanism of anti-foreign heredity substance, and it can destroy foreign heredity substance, for example nuclease.Yet it is more responsive that single stranded oligonucleotide and double chain oligonucleotide or oligonucleotide analogs compare described defense mechanism continually.Therefore an advantage is to use in a pair of oligonucleotide or the oligonucleotide analogs control agent or the genetic transcription of exsomatizing.

In addition, a pair of oligonucleotide of the present invention and/or oligonucleotide analogs can with two chains hybridization of goal gene, described goal gene can be when only hybridizing to a chain regulatory gene activity more strictly.

Because the melting temperature(Tm) of the crossbred of first sequence and second sequence is preferably lower than the crossbred between first sequence and the corresponding D NA and the melting temperature(Tm) of second sequence and corresponding D NA crossbred, so a pair of oligonucleotide analogs of intercalator pseudonucleus thuja acid that comprises the inventive method is preferably with gene and/or complementary gene sequence but not hybridization mutually.

Preferably comprise described oligonucleotide and/or oligonucleotide analogs, significantly be lower than such as the melting temperature(Tm) of the self-crossbred of first sequence and comprise that first sequence and described gene or its regulate the melting temperature(Tm) of the crossbred of the complementary strand of sequence or described gene or its complementary strand of regulating sequence.

Re-reading system can be arbitrary useful re-reading system, comprises two vitro system, system and stripped system in the body.For example re-reading system can be selected from yeast cell, fungal cell, mammalian cell, vegetable cell, bacterial cell, archeobacteria cell and virus.

Preferably, re-reading system is an intact cell.For example cell can be the human cell.Cell can be that isolated cells or cell can be included in live organism, in animal, people or plant materials.

Have the oligonucleotide and/or the nucleotide analog that reduce cross hybridization

For many purposes, preferably oligonucleotide and/or oligonucleotide analogs are only with target nucleic acid (RNA or DNA usually) but not hybridize with other homology complementary nucleic acid and nucleic acid analog that comprises described oligonucleotide itself.Especially not only β-homotype-the DNA that comprises probe, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-and TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA and PNA, and other nucleic acid and nucleic acid analog also have to the homology complementary nucleic acid of described probe same type or the trend of nucleic acid analog high affinity cross hybridization.

Usually need to use nucleic acid analog comparison homology complementary RNA or DNA target are had the more probe of high affinity.Yet a lot of known oligonucleotide analogs for example, but be not limited to β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-and TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA and PNA, comparison equates that homology complementary unmodified rna or DNA oligonucleotide probe have higher affinity to the homology complementary oligonucleotide analogue of same type.Therefore described oligonucleotide analogs can be stood on two complementary strands of same area that higher self-affinity and very difficult use hybridize to target duplex DNA nucleic acid at the same time.A problem in addition is to use the oligonucleotide analogs of respective complementary to himself.

Therefore, a purpose of the present invention provides oligonucleotide and/or the oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid, wherein significantly is lower than the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide and/or oligonucleotide analogs and homology complementary DNA (DNA crossbred) by described oligonucleotide and/or oligonucleotide analogs and the melting temperature(Tm) that comprises the homology complementary oligonucleotide of at least one intercalator pseudonucleus thuja acid and/or the duplex that oligonucleotide analogs is formed.

In one embodiment of this invention, a pair of homology complementary oligonucleotide and/or oligonucleotide analogs comprise at least one intercalator pseudonucleus thuja acid in each aforesaid oligonucleotide and/or oligonucleotide analogs, wherein said at least two intercalator pseudonucleus thuja acids location that is relative to each other, closely approaching each other during with convenient homology complementary oligonucleotide or oligonucleotide analogs hybridization.Preferably, at least 2 intercalator pseudonucleus thuja acids location that is relative to each other is opposite each other during to hybridization with convenient oligonucleotide analogs.More preferably, oligonucleotide or oligonucleotide analogs comprise above a pair of intercalator oligonucleotide, such as 2, for example 3, such as 4, for example, such as the intercalator pseudonucleus thuja acid that surpasses 5 pairs, every pair of intercalator pseudonucleus thuja acid location that is relative to each other wherein, at least two intercalator pseudonucleus thuja acids are closely approaching each other during with convenient oligonucleotide analogs hybridization.Preferably, each the intercalator pseudonucleus thuja acid in chain in a chain, be relative to each other the location during with convenient oligonucleotide or oligonucleotide analogs hybridization their two and two closely approaching each other.Most preferably, each intercalator in chain in a chain, be relative to each other the location during with convenient oligonucleotide or oligonucleotide analogs hybridization their two and two opposite each other.The result of this system will be a pair of homology complementary nucleic acid analogue that comprises two chains (chain 1 and chain 2), and its medium chain 1 has the affinity higher to complementary dna chain comparison chain 2, and chain 2 has the affinity higher to complementary dna chain comparison chain 1.Might produce a pair of probe like this, target sequence is compared its complementary probe chain for it and a pair of probe has higher affinity, and it can locate two chains of duplex DNA complementary region.The chain of probe centering can be directly or indirect detection.The result of this method will be to false negative and false positive tool low risk more, produces probe system to the better signal to noise ratio of target DNA thereby have higher sensitivity.Oligonucleotide and/or oligonucleotide analogs with oneself's hybridization level of reduction

In another preferred embodiment of the present invention, when intercalator pseudonucleus thuja acid embeds oligonucleotide and/or oligonucleotide analogs, can suppress or reduce cross hybridization basically self.

Therefore, an object of the present invention is to provide and comprise at least 2 oligonucleotide and/or the oligonucleotide analogs of intercalator pseudonucleus thuja acid as mentioned above, wherein said two intercalator pseudonucleus thuja acids location that is relative to each other is so that if they are closely approaching each other when oligonucleotide and/or oligonucleotide analogs oneself hybridization.

Preferably, if at least 2 intercalator pseudonucleus thuja acids are relative to each other the location so that opposite each other when oligonucleotide and/or oligonucleotide analogs oneself hybridization.More preferably, oligonucleotide and/or oligonucleotide analogs comprise above a pair of intercalator oligonucleotide, such as 2, for example 3, such as 4, for example, such as surpassing 5 pairs of intercalator pseudonucleus thuja acids, every pair of intercalator pseudonucleus thuja acid location that is relative to each other wherein, their two and two are closely approaching each other when hybridizing with convenient oligonucleotide analogs self.Preferably, every pair of intercalator pseudonucleus thuja acid is relative to each other opposite each other when locating with convenient oligonucleotide and/or oligonucleotide analogs oneself hybridization.In a preferred embodiment, described at least intercalator pseudonucleus thuja acid to one of fluorescent characteristics can be used as the signaling molecule of detection.

The melting temperature(Tm) that comprises the duplex between two homology complementary oligonucleotide analogue sequences of intercalator pseudonucleus thuja acid will depend on the number of intercalator pseudonucleus thuja acid of embedding and the position that has embedded described intercalator pseudonucleus thuja acid.Comparing melting temperature(Tm) with the identical oligonucleotide (analogue) that does not comprise arbitrary intercalator pseudonucleus thuja acid may reduce.Preferably, melting temperature(Tm) reduces at least 2 ℃, such as at least 5 ℃, and for example at least 10 ℃, such as at least 15 ℃, such as from 2 to 50 ℃, such as from 2 to 40 ℃, such as from 2 to 30 ℃, for example from 5 to 20 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, for example from 10 ℃ to 15 ℃, such as from 15 ℃ to 20 ℃, for example from 20 ℃ to 25 ℃, such as from 25 ℃ to 30 ℃, for example from 30 ℃ to 35 ℃, such as from 35 ℃ to 40 ℃, for example from 40 ℃ to 45 ℃, such as from 45 ℃ to 50 ℃.

In one embodiment of the invention, relate to suppress or reduce in a large number probe self-and/or cross hybridization, described probe comprises that one or more are selected from but are not limited to the Nucleotide of following Nucleotide: β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA or RNA.

In the present invention's one preferred embodiment, relate to suppress or reduce in a large number probe self-and/or cross hybridization, described probe comprises that one or more are selected from following Nucleotide: LNA, α-D-ribo-LNA, [3.2.1]-LNA, 2 '-R-RNA, 2 '-OR-RNA, α-D-RNA, β-D-RNA or RNA.

Therefore, a purpose of the present invention provides and comprises at least one oligonucleotide analogs of intercalator pseudonucleus thuja acid as mentioned above, significantly is lower than the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide analogs and oligonucleotide analogs when not comprising intercalator pseudonucleus thuja acid or homology complementary DNA (DNA crossbred) comprising the melting temperature(Tm) of described oligonucleotide analogs oneself crossbred.

Preferably, described oligonucleotide analogs is selected from: DNA, β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA and composition thereof, more preferably, oligonucleotide analogs can be selected from β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acyls are pressed-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA and the β-homotype-DNA that comprises significant quantity, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, the mixture of RNA or PNA.

The described melting temperature(Tm) that comprises oligonucleotide that at least one embeds the pseudonucleus thuja acid and/or the crossbred between oligonucleotide analogs and the DNA melting temperature(Tm) than the self-crossbred of described oligonucleotide analogs at least is high 2 ℃, such as at least 5 ℃, for example at least 10 ℃, such as at least 15 ℃, such as from 2 to 50 ℃, such as from 2 to 40 ℃, such as from 2 to 30 ℃, for example from 5 to 20 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, for example from 10 ℃ to 15 ℃, such as from 15 ℃ to 20 ℃, for example from 20 ℃ to 25 ℃, such as from 25 ℃ to 30 ℃, for example from 30 ℃ to 35 ℃, such as from 35 ℃ to 40 ℃, for example from 40 ℃ to 45 ℃, such as from 45 ℃ to 50 ℃, such as from 55 ℃ to 60 ℃, for example from 60 ℃ to 65 ℃.

In one embodiment of the invention, oligonucleotide analogs comprises the intercalator pseudonucleus thuja acid that at least one is above-mentioned, the location of wherein said intercalator pseudonucleus thuja acid is with relevant such as, but not limited to following non--DNA Nucleotide: β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA, perhaps other RNA quasi-molecule, therefore when oligonucleotide analogs hybridization or when self hybridizing, they are closely approaching each other.Preferably, the location of at least one intercalator pseudonucleus thuja acid is with relevant such as, but not limited to following non--DNA Nucleotide: β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-and TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA, perhaps other RNA quasi-molecule, therefore, when oligonucleotide hybridization or self hybridization intercalator pseudonucleus thuja acid relatively and between two non--DNA-quasi-molecules.

In addition, the present invention relates to design and Nucleotide that generation and the complementary non-intercalator pseudonucleus thuja acid of homology are modified or nucleotide analog are compared the method for the sequence that the solvent temperature of self crossbred reduces.Preferably, self crossbred comprises and is selected from following oligonucleotide or oligonucleotide analogs: low, medium or even high stringent condition under be tending towards the β-homotype-DNA of self hybridization, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA and composition thereof, described method comprises the steps:

A) the synthetic oligonucleotide analogs that has at least one intercalator pseudonucleus thuja acid, the modes of emplacement of described intercalator pseudonucleus thuja acid satisfy itself and at least one, and to be selected from following Nucleotide closely approaching: β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-and TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA, it is the part with described intercalator pseudonucleus thuja acid of following formula:

X-Y-Q

Preferably, intercalator pseudonucleus thuja acid is gone into mentioned above; And

Obtain described self hybridization when with oligonucleotide that does not comprise arbitrary intercalator pseudonucleus thuja acid or oligonucleotide analogs comparison for oligonucleotide analogs with lower melting temperature(Tm).

Melting temperature(Tm) can reduce according to the characteristic and the number of the intercalator pseudonucleus thuja acid that embeds, and according to the position that embeds described intercalator pseudonucleus thuja acid and under the situation of oneself's hybridization described embedding pseudonucleus thuja acid be positioned between Nucleotide and/or the nucleotide analog.Preferably, melting temperature(Tm) reduces at least 2 ℃, such as at least 5 ℃, and for example at least 10 ℃, such as at least 15 ℃, such as from 2 to 50 ℃, such as from 2 to 40 ℃, such as from 2 to 30 ℃, for example from 5 to 20 ℃, such as from 10 ℃ to 15 ℃, for example from 2 ℃ to 5 ℃, such as from 5 ℃ to 10 ℃, for example from 10 ℃ to 15 ℃, such as from 15 ℃ to 20 ℃, for example from 20 ℃ to 25 ℃, such as from 25 ℃ to 30 ℃, for example from 30 ℃ to 35 ℃, such as from 35 ℃ to 40 ℃, for example from 40 ℃ to 45 ℃, such as from 45 ℃ to 50 ℃.

Preferably at least 2 intercalator pseudonucleus thuja acids embed in described oligonucleotide or the oligonucleotide analogs when design oligonucleotides and/or oligonucleotide analogs.More preferably, locate them according to reducing oneself's hybridization as mentioned above.

More preferably, at least 2 intercalator pseudonucleus thuja acids import in described oligonucleotide or the oligonucleotide analogs, so that they and at least one are not limited to following Nucleotide or nucleotide analog located in connection: β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA, locator means satisfies closely approaching each other when oligonucleotide analogs is hybridized.More preferably, at least 2 intercalator pseudonucleus thuja acids import in described oligonucleotide or the oligonucleotide analogs, so as with they with at least 2 following Nucleotide located in connection: β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA, they are between described nucleotide analog when hybridizing to self with convenient oligonucleotide analogs.

Avoid the method for non-specific hybridization

An object of the present invention is to provide and reduce between oligonucleotide and/or the oligonucleotide analogs and the method for the nonspecific property hybridization of non-target nucleic acid and/or nucleic acid analog and/or oligonucleotide and/or oligonucleotide analogs.This is by providing one to comprise that the oligonucleotide and/or the oligonucleotide analogs of at least one intercalator pseudonucleus thuja acid are achieved.

In one embodiment of the invention, comprise the oligonucleotide of at least one intercalator pseudonucleus thuja acid and/or oligonucleotide analogs for complementary nucleic acid and/or nucleic acid analog comparison homology but not fully the complementary nucleic acid target have higher hybridization specificity.

In a preferred embodiment of the invention, comprise that for complementary nucleic acid and/or nucleic acid analog target comparison homology but not fully complementary nucleic acid and/or nucleic acid analog target are compared with homology complementary oligonucleotide that does not comprise arbitrary intercalator pseudonucleus thuja acid or oligonucleotide analogs and had higher hybridization specificity for the oligonucleotide of at least one intercalator pseudonucleus thuja acid and/or oligonucleotide analogs.

Therefore, under identical hybridization conditions, the described oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid is complementary fully but not exclusively complementary nucleic acid and/or nucleic acid analog with bound fraction, and described part is complementary fully significantly greater than by the described identical oligonucleotide that does not comprise arbitrary intercalator pseudonucleus thuja acid or oligonucleotide analogs bonded complementary fully but not exclusively complementary nucleic acid and/or nucleic acid analog.

Preferably, comprise that the described oligonucleotide analogs of at least one intercalator pseudonucleus thuja acid will be in conjunction with obviously more most nucleic acid and/or nucleic acid target but not non-target nucleic acid and/or nucleic acid analog.

Detect single nucleotide polymorphism (SNP)

Relate to the detection of SNP in one embodiment of the invention, promptly detect with from the different nucleic acid target of another nucleotide sequence.The probe that utilization is made up of oligonucleotide that contains at least one intercalator pseudonucleus thuja acid and/or oligonucleotide analogs is implemented to detect.Detection based on probe to the duplex and the probe of complementary target sequence and comprise the difference of melting temperature(Tm) between the duplex of target sequence of at least a SNP.Preferably, when with the melting temperature(Tm) of described oligonucleotide or oligonucleotide analogs with they complementary target and and described to the SNP sequence oligonucleotide or have higher melting temperature(Tm) than oligonucleotide that does not comprise described intercalator or oligonucleotide during the oligonucleotide analogs comparison.In a preferred embodiment, described oligonucleotide analogs comprises at least one intercalator pseudonucleus thuja acid.In another preferred embodiment, described intercalator pseudonucleus thuja acid is closely approaching with the incomplementarity nuclear base of the duplex that comprises described probe and described SNP target nucleic acid and/or nucleic acid analog and/or oligonucleotide and/or oligonucleotide analogs.

In another preferred embodiment of the present invention, described oligonucleotide and/or oligonucleotide analogs comprise at least two intercalator pseudonucleus thuja acids, the position satisfy when described probe hybridization during to the sequence that comprises SNP at least one intercalator pseudonucleus thuja acid be positioned at the downstream that upstream and at least one intercalator pseudonucleus thuja acid are positioned at least one base mispairing.

In the present invention's one more preferred, described intercalator pseudonucleus thuja acid is positioned at 4 nuclear bases of the every side of described incomplementarity nuclear base of described crossbred, and the formation of described crossbred is because the hybridization between described probe and the described SNP that comprises complementary sequence.

Most preferably described intercalator pseudonucleus thuja acid is positioned at 2 nuclear bases of the every side of described incomplementarity nuclear base of described crossbred, and the formation of described crossbred is because the hybridization between described probe and the described SNP that comprises complementary sequence.

In multiple analysis, adjust the method for melting temperature(Tm)

An object of the present invention is when identical reaction vessel or general surface and/or can the condition of stdn experiment condition under when carrying out, the method of adjusting the melting temperature(Tm) difference that runs in the multiple crossing analysis is provided, especially consider temperature and/or buffer condition to improve hybrid experiment exactness relatively, described experiment condition is the condition that hybrid experiment carries out.These purposes are important for the most of nucleic acid based on screening, because this class screening method is almost specially based on nucleic acid hybridization.Present in addition this screening is in order to increase the level of multiple method.In this analysis, consider hybridization, the application limitations of selecting comparable nucleotide sequence has been applied many variability to not homotactic melting temperature(Tm).

Therefore, one aspect of the present invention just provides and is used for the system that multiple crossing is analyzed as mentioned above.Described system can be arbitrary suitable device, such as chamber or kapillary or arbitrary other flow control apparatus in array, chip, electronic chip, the reaction vessel, glass capillary, flow cytometer, at least one oligonucleotide and/or oligonucleotide analogs have wherein been arranged such as the microtiter plate micropore.

Because the A-T base pair only has two hydrogen bonds, and the G-C base pair has the fact of three hydrogen bonds and provides and the target that is rich in AT is had the oligonucleotide of strong hybridization affinity and/or oligonucleotide analogs will remedy the nucleic acid that every nuclear base pair provides by A-and T-nuclear base and the low melting temperature(Tm) of nucleic acid analog.

Therefore one aspect of the present invention provides and has the oligonucleotide that comprises at least one intercalator pseudonucleus thuja acid of the present invention and/or the system of oligonucleotide, will stablize described oligonucleotide and/or oligonucleotide analogs and homology complementary nucleic acid or nucleic acid analog so wherein said intercalator owing to embed is next at least the AT base pair.

Another aspect of the present invention provides the oligonucleotide analogs with about identical nuclear base number, described nuclear base comprises at least one intercalator pseudonucleus thuja acid of the present invention, and wherein the hybridization affinity that the nucleic acid of AT and/or nucleic acid analog are rich in complementation to homology can be compared with the hybridization affinity to the target that is rich in GC.

Equally, a purpose of the present invention provides the method for adjusting melting temperature(Tm) during multiple crossing is analyzed between different IPs thuja acid and/or nucleic acid analog sequence and their homology complementary nucleic acid target and/or the nucleic acid analog target, wherein oligonucleotide and/or oligonucleotide analogs sequence comprise at least one intercalator pseudonucleus thuja acid, and the melting temperature(Tm) of wherein said oligonucleotide analogs sequence and described nucleic acid and/or nucleic acid analog is than the melting temperature(Tm) of the described oligonucleotide analogs sequence that does not comprise intercalator pseudonucleus thuja acid and described homology complementary nucleic acid target or nucleic acid analog target homology more significantly.

In one embodiment of the invention, the oligonucleotide analogs that comprises at least one intercalator pseudonucleus thuja acid has one or more intercalator pseudonucleus thuja acid, the zone that specificity embedding homology complementary nucleic acid or nucleic acid analog are rich in A-and/or T-after hybridization.

In another embodiment of the present invention, the described described oligonucleotide analogs position that comprises at least one intercalator pseudonucleus thuja acid is satisfied embed the zone that described homology complementary nucleic acid or nucleic acid analog are rich in A-and/or T-after hybridization, the location specific ground of described one or more intercalator pseudonucleus thuja acid increase power to be rich in A-and/or T-target in conjunction with affinity.

If provide in the hybridization of oligonucleotide that the different homology complementary nucleic acid that surpasses one or nucleic acid analog detect simultaneously to needs or oligonucleotide analogs, the hybridization of described oligonucleotide and/or oligonucleotide analogs and described homology complementary nucleic acid and/or nucleic acid analog need be carried out usually under the same conditions.Therefore, an advantage is that described crossbred has the melting temperature(Tm) that approximately equates.

In a preferred embodiment of the invention, at least two oligonucleotide or oligonucleotide analogs are provided, wherein provide at least one to comprise at least one intercalator pseudonucleus thuja acid of the present invention, be used at least two different homology complementary nucleic acid or nucleic acid analogs of multiple detection.In another preferred embodiment, the melting temperature(Tm) of the crossbred between at least two described oligonucleotide or oligonucleotide analogs and described homology complementary nucleic acid or the nucleic acid analog is very low.Preferred, all described crossbreds have similar melting temperature(Tm), and such as substantially the same, that is, the difference between the melting temperature(Tm) is less than 5 degrees centigrade, more preferably less than 3 degrees centigrade.

By intercalator pseudonucleus thuja acid preferably being assigned into the zone that described oligonucleotide or oligonucleotide analogs are rich in A-and/or T-, thus adjust usually by be rich in the nucleic acid of hybridization and/or the nucleic acid analog duplex structure less intensity for hybridization that A-and/or T-zone provide and therefore described nucleic acid or nucleic acid analog target to the hybridization temperature of the described oligonucleotide analogs of complementary basically more similar in appearance to not with the pseudonucleus thuja acid of intercalator.

In one embodiment of the invention, the multiple crossing system comprises at least 1, such as from 2 to 10 ⁶, such as from 2 to 10 ⁵, such as from 2 to 10 ⁴, such as from 2 to 10 ³Such as from 2 to 5, for example from 5 to 10, such as from 10 to 50, for example from 50 to 100, such as from 100 to 1000, for example from 1000 to 5000, such as from 5000 to 10000, for example from 10000 to 50000, such as from 50000 to 100000, the different sequences of from 100000 to 1000000 oligonucleotide that the present invention may provide and/or oligonucleotide analogs for example, wherein at least one oligonucleotide and/or oligonucleotide analogs comprise at least one intercalator pseudonucleus thuja acid, and preferably all oligonucleotide and/or oligonucleotide analogs comprise at least one intercalator pseudonucleus thuja acid.

In another embodiment of the present invention, can provide at least 1, such as between 2 and 5, for example between 5 and 10, such as between 10 and 50, for example between 50 and 100, such as between 100 and 1000, for example between 1000 and 5000, such as between 5000 and 10000, for example the different sequences of an oligonucleotide analogs of the present invention are attached on the solid support between 10000 and 50000.

The method of stable oligonucleotide of nuclease and/or oligonucleotide analogs is provided

One aspect of the present invention relates to stable oligonucleotide of nuclease and/or oligonucleotide analogs.This comprises that by providing at least one oligonucleotide and/or oligonucleotide analogs that embeds embedding pseudonucleus thuja acid of the present invention is achieved.

The oligonucleotide and/or the nucleotide analog that comprise at least one pseudonucleus thuja acid can be used in the great majority analysis, the wherein nuclease-mediated oligonucleotide and/or the decomposition of oligonucleotide analogs can cause non-optimal results, for example the probe analysis that in viable cell, in the DNA cloning process, carries out or, wherein use archaeal dna polymerase with exonuclease activity.

The separation sequence specific DNA

The invention provides from the method for the mixture separation sequence specific DNA that comprises nucleic acid, described method comprises the steps:

A) provide the mixture that comprises nucleic acid; And

B) provide one or more different oligonucleotide or oligonucleotide analogs, wherein the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and homology complementary DNA (DNA crossbred) is significantly higher than the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and homology complementary RNA (RNA crossbred), and wherein said oligonucleotide or oligonucleotide analogs can with described sequence specific DNA hybridization (target DNA); And

C) with described mixture and described oligonucleotide or oligonucleotide analogs can with incubation under the condition of homology complementary oligonucleotide or oligonucleotide analogs hybridization; With

D) from mixture separation oligonucleotide or oligonucleotide analogs together with the nucleic acid that hybridizes to described oligonucleotide or oligonucleotide analogs;

Therefore obtain isolating sequence specific DNA and the isolating remaining mixture that comprises nucleic acid.

The mixture that comprises nucleic acid can comprise arbitrary nucleic acid or nucleic acid analog, and for example it can include but not limited to: DNA, β-homotype-DNA, β-D-pyrans altrose base-NA, β-D-glucopyranosyl-NA, β-D-Allopyranusyl-NA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, α-D-RNA, β-D-RNA, RNA or PNA.Preferably nucleic acid is RNA and DNA.

The mixture that comprises nucleic acid for example can be to comprise that the intact cell of nucleic acid or mixture can be the cell extracts that comprises nucleic acid.Mixture can be the nucleic acid of purifying in addition, mixture can be that the synthetic preparation mixture of nucleic acid or mixture can be chemically or the mixture of the nucleic acid of enzymatically modifying or nucleic acid analog, for example the DNA that transforms of bisulfate or the DNA of part restriction enzyme enzyme liberating.

With being to be selected from the oligonucleotide that comprises at least one aforesaid intercalator pseudonucleus thuja acid or the oligonucleotide or the oligonucleotide analogs of oligonucleotide analogs from the employed preferred oligonucleotide of method of the mixture separation sequence specific DNA that comprises nucleic acid or oligonucleotide analogs.

This oligonucleotide or oligonucleotide analogs can comprise from 5-100, such as from 5-50, such as from 5-30, such as from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as from 20 to 30, for example from 30 to 100 Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.Preferably, described oligonucleotide or oligonucleotide analogs comprise from 10 to 50 Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.

Might surpass a kind of sequence specific DNA from described mixture separation.In order to do like this, different oligonucleotide or oligonucleotide analogs must be provided, wherein at least a oligonucleotide that provides or oligonucleotide analogs can be hybridized with each sequence specific DNA.In this embodiment, can use the multiple crossing system, described optimum system choosing ground comprises at least 1, such as from 2 to 10 ⁶, such as from 2 to 10 ⁵, such as from 2 to 10 ⁴, such as from 2 to 10 ³Such as from 2 to 5, for example from 5 to 10, such as from 10 to 50, for example from 50 to 100, such as from 100 to 1000, for example from 1000 to 5000, such as from 5000 to 10000, for example from 10000 to 50000, such as from 50000 to 100000, for example from 100000 to 1000000 the present invention different oligonucleotide that may provide and/or the sequence of oligonucleotide analogs.

Carrying out this method is in order to realize one of several objects.For example, described target can be from comprising that RNA with identical or substantially the same sequence and the mixture of DNA remove sequence specific DNA.Identical sequence is understood as that Nucleotide T that DNA comprises is arranged in the site of RNA Nucleotide U, and other sequence is identical.RNA that sequence specific DNA has been removed and the mixture of DNA for example can be used as the template that is used for inverse transcription polymerase chain reaction (RT-PCR) and have and remove because the caused false positive of appearance that DNA pollutes.A common problem is exactly the RNA sample that obtains not contain DNA.For example Ambion company is described in its 6th page of 2001 catalogue: how normally used a large amount of RNA method of purifications produce the RNA prepared product that does not contain DNA.In the prior art, can handle by deoxyribonuclease and remove the DNA pollution, described processing is expensive and time-consuming because need to remove deoxyribonuclease usually after processing.

The invention provides the method for removing sequence specific DNA from the RNA sample.The known arbitrary method isolation of RNA of person skilled in the art can be passed through, especially when from the compound bio sample, isolating RNA, described method can be utilized.

For example total RNA can separate by several method according to routine, and described method comprises based on guanine thiocyanate-/acid phenol: the method for chloroform, and based on filtering bonded method and the method by the CSCl gradient centrifugation.No matter which kind of method is polluted chromosomal DNA and is present in (

Ambion TechNotes

7,1,2000) in the isolating RNA sample usually, it may cause problem in the method that can use RNA.For the introduction of polluting relevant problem with the DNA of isolation of RNA for example referring to the critical factor of PCR, the 18th chapter, Qiagen, www.qiagen.com.Owing to comprise the oligonucleotide of intercalator pseudonucleus thuja acid of the present invention or the selectivity characrerisitic of oligonucleotide analogs, might rapidly and remove the DNA pollutent easily and not excessive loss RNA material.000

Therefore preferably, comprise that the remaining mixture of nucleic acid is substantially free of sequence specific DNA.Therefore, preferably should provide enough oligonucleotide or oligonucleotide analogs, so that every kind of sequence specific DNA molecule can be hybridized with a kind of oligonucleotide or oligonucleotide analogs.

In another embodiment of the present invention, described method relates to the method for removing the DNA pollutent from the sample of the polyadenylic acid that comprises RNA (mRNA).Combine the polyadenylic acid that comprises RNA according to conventional purifying based on its specificity with poly-(DT) (including only the DNA oligonucleotide of thymidine) or poly-(U) (including only the RNA oligonucleotide of uridine).The post that for example has the oligonucleotide (U) that is attached to cellulosic oligonucleotide (DT) or is attached to sephadex usually is used for mRNA purification process (molecular biology current approach (CurrentProtocols in Molecular Biology), 1995, John Wiley ﹠amp; Sons Inc.USA, Chap.4.5).Yet the poly+RNA of purifying usually comprises the DNA pollutent, especially it usually comprise can with poly-dT and/or oligonucleotide (U) bonded DNA.In order to obtain not contain the mRNA of DNA pollutent, therefore need carry out the selection of two-wheeled poly-(DT).This usually is labor-intensive for routine analysis.Also can change the relative content of independent transcript in addition, may be since between the tissue discrepant polyadenylation or in response to biology stimulate (Ambion Technical bulletin 176, www.ambion.com).The present invention relates to utilize above-mentioned method to remove the DNA method of depolluting, wherein oligonucleotide or oligonucleotide analogs are made up of intercalator pseudonucleus thuja acid and thymidine Nucleotide or nucleotide analog basically.

In another embodiment, thus in fact can hybridize to the oligonucleotide of a restriction group or oligonucleotide analogs by sequence-specific removes all genomic dnas in the nucleic acid mixture.Run through the be scattered here and there repetitive sequence of 100 to 500 dissimilar base pairs of mammalian genes group, wherein the abundantest is SINES (repeat short the dispersion) and LINES (long disperse repetition).Especially Alu system reappears in the people's gene group about 1,000,000 times corresponding to the average repetition once of per 3000 bases.

An object of the present invention is to provide the optionally selection of the RNA/DNA of oligonucleotide or oligonucleotide analogs sequence of RNA that difference preferably includes intercalator pseudonucleus thuja acid of the present invention, wherein said sequence comprises a large amount of eukaryotes and the known repeat element of mammalian genes group preferably.Therefore, when the nucleic acid mixture in eukaryote or Mammals source is provided, preferably by the restricted degradation of dna of enzyme, when the method for the broken nucleic acid of the known arbitrary other parts of supersound process or person skilled in the art is carried out, the RNA selectivity sequence of described selection will hybridize to the described repeat element in the genomic dna of described eukaryote or mammalian nucleic acid mixture under suitable stringent condition.Describedly have if provide to separate, can separate remaining RNA and therefore can the described RNA of purifying from described genomic dna in conjunction with the oligonucleotide of DNA or the solid support of oligonucleotide analogs with the described oligonucleotide of solid support bonded or oligonucleotide analogs sequence and from described nucleic acid mixture.

The RNA mixture that acquisition is substantially free of contaminating dna provides the selection that difference preferably includes the RNA/DNA of the RNA-selectivity oligonucleotide of intercalator pseudonucleus thuja acid of the present invention or oligonucleotide analogs sequence to another approach, and wherein said sequence has a certain length and forms irregular distribution to comprise all possible sequence of this length.Therefore the selection of described oligonucleotide or oligonucleotide analogs sequence can with by described all represented homology complementary dna sequences hybridization of sequence that form irregular distributed collection.Describedly have if provide to separate, can separate remaining RNA and therefore can the described RNA of purifying from described genomic dna in conjunction with the oligonucleotide of DNA or the solid support of oligonucleotide analogs with the described oligonucleotide of solid support bonded or oligonucleotide analogs sequence and from described nucleic acid mixture.

An object of the present invention is above-mentioned two purposes combination, described target preferably includes the selection of the RNA/DNA of the RNA-selectivity oligonucleotide of intercalator pseudonucleus thuja acid of the present invention or oligonucleotide analogs sequence for difference is provided, and described RNA-selectivity oligonucleotide or oligonucleotide analogs are represented the sequence of one group of repeat element or stochastic distribution.The combination of two targets can these two kinds of methods of balance shortcoming separately.

Another target of this method may be that the mixture from DNA and RNA obtains sequence specific DNA in an one-step method, and described method does not comprise by for example using RNase (rnase) to handle the process of further removing sequence-specific RNA.

For example the method for the present invention's description can be used for from the DNA sample of compound bio sample or sample separation purifying.Therefore described method can for example be used for from the DNA of cytolysis compound bio mixture separation biological mixture such as the cell release that comprises nucleic acid, and perhaps DNA can be the DNA from quite pure sample purifying.This method can for example be included in lysing cell in the hybridization medium that comprises strong chaotropic agent, and contacting split product also with oligonucleotide or oligonucleotide analogs under hybridization conditions, separation sequence is used for further application.

Separated DNA for example can be used for the clone, and the template as amplified reaction is used for hybridization analysis, is used for diagnosis or the known method of arbitrary other person skilled in the art.

Can the known arbitrary mode of person skilled in the art the mixture of nucleic acid and described oligonucleotide or oligonucleotide analogs be used to carry out incubation under the condition that hybridizes to.

In one embodiment of the invention, nucleic acid mixture will comprise chaotropic agent, target nucleic acid and be complementary to the oligonucleotide or the oligonucleotide analogs of target nucleic acid substantially.Preferably, before hybridization or simultaneously nucleic acid mixture can be heated to the temperature of destruction albumen/nucleic acid interaction, so that the hybridization between maximization oligonucleotide or oligonucleotide analogs and the target thereof.

For example, nucleic acid mixture can be heated to the interactional temperature of destruction albumen/nucleic acid and be cooled to oligonucleotide subsequently or oligonucleotide analogs and target DNA between hybridize.

When using high affinity oligonucleotide or oligonucleotide analogs, can under elevated temperature, hybridize, preferably temperature should completely destroy DNA:DNA and the interaction of DNA:RNA.Preferably, temperature is low hybridizes to being enough between oligonucleotide or oligonucleotide analogs and target DNA (DNA crossbred).

Because the melting temperature(Tm) of DNA crossbred preferably is higher than the homology complementary DNA: the melting temperature(Tm) height of dna double spiral and RNA crossbred, the double-helical hybridization of RNA crossbred or DNA:DNA so the selection of temperature should be satisfied the DNA crossbred.

Also can select further feature except that melting temperature(Tm) to optimize the specific hybrid of DNA crossbred, for example pH of salt concn and/or damping fluid.

Can use diverse ways from oligonucleotide or the oligonucleotide analogs of mixture separation, preferably hybridize to the DNA of described oligonucleotide together with nucleic acid.For example can be by gel electrophoresis, the known arbitrary method of gel-filtration or person skilled in the art is separated.

In a preferred embodiment of the invention, oligonucleotide or oligonucleotide analogs are attached on the solid support.Then can by from the described solid support of mixture separation from the mixture separation oligonucleotide or oligonucleotide analogs together with the nucleic acid that hybridizes to described oligonucleotide.

The multiple solid support of result according to expectation is applicable to described method.

Solid support is an activated surface in one embodiment.Activated surface promotion oligonucleotide or oligonucleotide analogs combine with solid support.

For example solid support can be selected from the encoded beads of magnetic bead, metallic bead, the pill of aluminium, sepharose 4B, sepharose pearl, arbitrary kind, for example bar code pearl, glass, plastic surface, heavy metal and chip surface.

Magnetic bead comprises the pearl that contains magnetic material, and it can utilize magnetic to separate pearl from suspension.

The agarose pearl for example can separate by centrifugal or filtration from suspension with the sepharose pearl.

Plastic surface comprises microtiter plate for example or other is applicable to for example plastic device of diagnosis.Chip surface can be made by arbitrary suitable material, for example sheet glass, resin board, metal sheet, with polymer-coated sheet glass, with the sheet glass of metal plating coating and with the resin board of metallic coating coating.Also can use SPR (surface plasmon resonance) sensor board, it is described in Japanese Patent and discloses among the 11-332595 temporarily.Also can use CCD, for example be described in Nucleic Acids Research, 1994, Vol.22, No.11 is among the 2124-2125.

Chip surface comprises the little polyacrylamide gel on the sheet glass, can oligonucleotide or oligonucleotide analogs be fixed (Yershov, people such as G., Proc Natl.Acad.Sci.USA on it by between polyacrylamide and oligonucleotide, forming covalent linkage, 94,4913 (1996).

Chip surface also can be Sosnowski, and R.G. waits the people, Proc.Natl.Acad.Sci.USA, 94, the silicon that 1119-1123 (1997) describes.This chip is prepared by the method that comprises the steps: row's microelectrode is placed on the silicon, on microelectrode, form the agarose layer that comprises Streptavidin, and by the agarose layer positively charged is adsorbed onto the dna fragmentation of biotin modification on the agarose layer.

In addition, chip surface can be as Schena, and M. waits people Proc.Natl.Acad.Sci.USA, 93, described being prepared of 10614-10619 (1996), wherein said method comprise the steps: to prepare the suspension of amido modified PCR product in SSC (that is standard sodium-chlor-citric acid solution), with the suspension point sample on slide glass, the slide glass of insulation point sample is handled the slide glass of incubation with sodium borohydride, and heats the slide glass of handling like this.

Here the method for Miao Shuing has other advantage, because they only need the sample and the analytical reagent of minimum treat amount.Therefore can provide the ready-made reagent solution that can use, for example, this ready-made reagent solution that can use can comprise chaotropic agent, other suitable component such as damping fluid or washing composition, can with can with the solid support bonded oligonucleotide or the oligonucleotide analogs of target nucleic acid hybridization.

Easily, thus sample for example the compound bio sample can be directly combine and can hybridize once going on foot with the pre-preparation reagent of hybridization.The solution of combination can cool off up to hybridizing then as the temperature that needs that is heated to described here.The hybridization complex that simple then washing produces is with the material of removing not hybridization and the degree of determining hybridization.

Detect the method for sequence specific DNA

One aspect of the present invention relates to the method that detects sequence specific DNA (target DNA) in the mixture that comprises nucleic acid and/or nucleic acid analog, and described method comprises the steps:

A) provide nucleic acid mixture; And

B) provide one or more different oligonucleotide or oligonucleotide analogs, be significantly higher than the melting temperature(Tm) of the crossbred (RNA crossbred) that comprises described oligonucleotide or oligonucleotide analogs and homology complementary RNA comprising the melting temperature(Tm) of the crossbred (DNA crossbred) of described oligonucleotide or oligonucleotide analogs and homology complementary DNA, and wherein said oligonucleotide or oligonucleotide analogs are complementary to described sequence specific DNA (target DNA) substantially; And

C) described mixture and described oligonucleotide or oligonucleotide are incubated under hybridization conditions; And

D) detect oligonucleotide or the oligonucleotide analogs that hybridizes to sequence specific DNA.

The advantage that described method surpasses method known in the state of the art is that this method can sequence-specific detects the DNA in the mixture that comprises described sequence specific DNA with similar sequences and RNA.Therefore, handle and significantly to reduce without rnase from the background signal of RNA.

Preferably mixture comprises DNA and RNA.More preferably mixture does not comprise other nucleic acid or the nucleic acid analog except that DNA and RNA.

Comprise that with detection the employed preferred oligonucleotide of method of sequence specific DNA in the mixture of nucleic acid and/or nucleic acid analog or oligonucleotide analogs are to be selected from the oligonucleotide that comprises at least one aforesaid intercalator pseudonucleus thuja acid or the oligonucleotide or the oligonucleotide analogs of oligonucleotide analogs.

This oligonucleotide analogs for example can comprise 3 to 10, such as 10 to 15, and for example 15 to 20, such as 20 to 30, for example 30 to 100 Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.Preferably described oligonucleotide analogs comprises Nucleotide and/or nucleotide analog and/or the intercalator pseudonucleus thuja acid between 3 to 50.

And oligonucleotide analogs can be arbitrary oligonucleotide analogs of the intercalator pseudonucleus thuja acid that comprises that the present invention describes.

Mixture can be included in the cell, for example in the intact cell.Cell for example can be prokaryotic cell prokaryocyte or eukaryotic cell, such as vegetable cell or mammalian cell.In such embodiment, described method can be used in situ hybridization.

Described method can comprise the separating step before detecting, wherein Za Jiao oligonucleotide or the oligonucleotide analogs oligonucleotide or the oligonucleotide analogs of never hybridizing separated, and described step can promote the specific hybrid of a hybridization oligonucleotide or oligonucleotide analogs.For example, the mixture of nucleic acid can be fixed on the solid support before hybridizing with oligonucleotide or oligonucleotide analogs.After the hybridization, Za Jiao oligonucleotide or oligonucleotide analogs can not rinse out and can detect the oligonucleotide or the oligonucleotide analogs of hybridization.

Perhaps, described method can comprise from the method for aforesaid mixture separation sequence specific DNA before detecting.For example, can be rinsed after oligonucleotide or oligonucleotide analogs can and be hybridized uncombined nucleic acid in conjunction with solid support and can detect bonded nucleic acid.

For example target DNA for example is a special genes, gene segment, little satellite-ellite or arbitrary other dna sequence dna.The particularly importantly detection of specific DNA, described DNA can be Eukaryotic, protokaryon, archeobacteria or viral source.Importantly be that the present invention can help to diagnose various transmissible diseases by analyzing the known specific sequence relevant with specific microorganism.Target DNA can be provided in the compound bio mixture of nucleic acid (RNA, DNA and/or RRNA) and non-nucleic acid, for example the thick extract of an intact cell or cell.

If target DNA is double-stranded or has significant secondary and tertiary structure on the contrary that they may heat before hybridization.In this case, heating can occur in and introduce nucleic acid to the hybridization medium that comprises oligonucleotide or oligonucleotide analogs before or afterwards.Sometimes also may be before hybridization analysis from compound bio sample extraction nucleic acid to reduce background interference by arbitrary methods known in the art.Double-stranded target DNA also can detect by triple forms and/or the chain intrusion of discussing here.

Hybridization of the present invention and extraction method can be used for the compound bio mixture of nucleic acid (DNA and/or RNA) and non-nucleic acid.This compound bio mixture comprises various eukaryotes and prokaryotic cell prokaryocyte, comprises protoplastis; Perhaps other biomaterial of target thymus nucleic acid that can berth.Therefore described method can be used for animal and people's cell of tissue culture, from for example blood, serum, blood plasma, netted red blood corpuscle, lymphocyte, urine, myeloid tissue, the animal of celiolymph and people's cell or organize examination of living tissue (for example from blood or lymph or any, the muscle biopsy of homogenate in lysis buffer, liver biopsy, renal biopsy, biopsy of urinary bladder, biopsy of bone, the cartilage examination of living tissue, skin biopsy, the pancreas examination of living tissue, the enteron aisle examination of living tissue, biopsy of thymus, breast, the uterus examination of living tissue, biopsy of testis, eyes examination of living tissue, perhaps big biosy of brain tissue) the next arbitrary product of preparation, vegetable cell or other are to responsive cell and the bacterium of infiltration concussion, the zymic cell, virus, mycoplasma, protozoon, Rickettsiae, fungi and other little microbial cell or the like.The described mixture that comprises nucleic acid also can be used for deriving from or extract the sample from food, beverage, water, medicament, private management article, milk-product or environmental sample.Analysis of the present invention and separation method can be used for for example detecting the non-pathogenic or pathogenic microorganism of target.Comprise the specific hybrid between the nucleic acid that exists in the oligonucleotide of intercalator pseudonucleus thuja acid or oligonucleotide analogs and the biological sample by detection, can determine the existence of microorganism.

The solution that comprises high concentrations of guanidine, guanine thiocyanate-or some other chaotropic agent and washing composition is cracking protokaryon and eukaryotic cell effectively, and carries out the specific hybrid of the interior source DNA of oligonucleotide analogs of the present invention and release simultaneously.Solution needn't comprise outer arbitrary other component of damping fluid commonly used and washing composition to promote cracking and the dissolving and the nucleic acid hybridization of cell.

If before hybridization, use extracting method, can be used in the employed technology of isolating nucleic acid such as the organic solvent of phenylic acid and chloroform.Traditionally, be separated such as the organic solvent utilization of phenol or phenylic acid-chloroform combination and be used for extracting nucleic acid (Ausubel etc. are at CurrentProtocols in Molecular Biology, pub.John WILEY ﹠amp; Sons (1998).These methods can effectively be used with lysate of the present invention, yet the advantage of the inventive method is not need loaded down with trivial details extracting method, have therefore improved the performance of high throughput analysis.Preferably, lysis buffer/hybridization substratum will comprise standard buffer solution and washing composition to promote the cracking of cell, still can comprise effective hybridization of the oligonucleotide analogs of intercalator pseudonucleus thuja acid simultaneously.Can use all 0.05 Trisodium Citrate, Tris-Tris-HCl according to appointment, PIPES or HEPES, preferably Tris-HCl damping fluid to 0.1M concentration.Hybridization medium preferably will also comprise about 0.05 to 0.5% ion or nonionic detergent, such as sodium laurylsulfonate (SDS) or Sarkosyl (Sigma Chemical Go., St.Louis, Mo.) and 1 and 10mM between EDTA.The additive that also can comprise other, such as the volume-exclusion agent that comprises various polarity water soluble or inflatable dose, such as anion pp acid esters or polymethacrylate, and charged glycopolymers, such as asuro etc.The specificity of hybridization or severity can be for example be controlled by concentration that changes chaotropic agent and the concentration of type and NaCl, and NaCl concentration is generally 0 and 1M NaCl, such as 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 or 1.0M NaCl between.

Destroy the chaotropic agent of albumen secondary and tertiary structure, for example such as the guanidinesalt and the thiocyanate-(GnSCN) of Guanidinium hydrochloride (GNHCI), perhaps urea, lithium chloride and other thiocyanate-s can be used in combination with the nucleic acid that exists of separating natural with washing composition and reductive agent such as beta-mercaptoethanol or DTT and suppress nuclease.The application of chaotropic agent in nucleic acid extraction and hybridization is described in EP and discloses in 0,127 327, is incorporated herein by reference herein.

In crossover process, will provide and be complementary to target DNA substantially, comprise the oligonucleotide analogs of intercalator pseudonucleus thuja acid.

In order to detect the oligonucleotide analogs that comprises intercalator pseudonucleus thuja acid, they can be connected to (biological example element, fluorescein, magnetic corpuscular or the like) on the group.Perhaps they can for good and all combine with solid phase or particle in advance, for example carry out (WO 96/31557) by the anthraquinone photochemical method.

The attracting possibility of the present invention is to utilize at not homotactic different oligonucleotide analogs in the genome, described different oligonucleotide analogs point sample also for good and all is being attached to (Nature Genetics on the surface on the array, suppl.vol.21, January 1999,1--60 and WO96/31557).This subsequently array can be incubated with the mixture of the lysis buffer/hybridization medium that comprises dissolved cell and many suitable detection oligonucleotide or oligonucleotide analogs.So cracking and hybridization will take place and can wash array at last and suitably expansion.The result of this method will be the semi-quantitative assessment of a large amount of different target DNAs.

For DNA or RNA, need will change with the change of hybridization medium and/or washing substratum severity with the complementary degree that oligonucleotide or oligonucleotide analogs form stable hybridization complex (duplex).Complementary nucleic acid may reside in the pre-preparation hybridization medium or some time points introducings after a while before hybridization.

Hybridization medium mixes with biological sample with cracking that promotes cell and nucleic acid pairing.Preferably, the amount that joins the biological sample in a large amount of hybridization mediums is with not too large, and the volume ratio of biological example sample volume and hybridization medium can be about 1: 10.

In one embodiment of the invention, hybridizing method of the present invention preferably carries out in mixture biological sample previous step.Yet, can consider under certain conditions small machinery or other processing.For example, may wish before hybridizing such as extracting nucleic acid with the clarification lysate by low-speed centrifugal or filtration or before hybridizing as mentioned above.

Hybridization analysis of the present invention can or be similar to the method for immunity that instructs given here by arbitrary method well known by persons skilled in the art to carry out.Preferred analytical procedure is sandwich analysis and change and competition or substitutability analysis.Hybridization technique is generally described in " nucleic acid hybridization, application method " ED.Hames, B.D. and Higgins, S.J., IRL Press, 1985; Gall and Pardue (1969), Proc.Natl.Acad.Sci., U.S.A., 63:378-383; And John, Burnsteil and Jones (1969) Nature is among the 223:582-587.Further improving of hybridization technique is known and can uses easily those skilled in the art.

In one embodiment of the invention, comprise that the oligonucleotide of intercalator pseudonucleus thuja acid or oligonucleotide analogs are as the capture probe in analyzing.Preferably described capture probe is attached to solid surface, and the surface of droplet price fixing micropore for example is on chip surface or the microballon.Therefore can carry out easily and thereby very effective washing process makes the reaction based on various enzymatics that can increase the invention performance become possibility.The most significant is that the sensitivity of hybridization analysis can be strengthened by using the nucleic acid amplification system, the described amplification system target DNA to be detected that increased.The example of this system comprises polymerase chain reaction (PCR) system, isothermal duplication and ligase chain reaction (LCR) (LCR) system.Other method known to those skilled in the art is such as being amplification (NASBA based on nucleotide sequence ^TM, Cangene, Mississauga, Ontario) and Q β replicative enzyme system.PCR is the archaeal dna polymerase primer extension method that relies on the repetition DNA selection sequence of template.The use that described method depends on excessive Auele Specific Primer is duplicated succeeded by the extension step of sex change and polysaccharase repeatedly with the archaeal dna polymerase that opens beginning DNA polynucleotide specific sub sequence.The PCR system is (referring to a United States Patent (USP) 4,683,195 and United States Patent (USP) 4,683,202) known in the art.For the out of Memory of considering PCR method referring to PCR application manual (PCR Applications Manual) second edition.RocheDiagnostics?or?PCR?Protocols：A?Guide?to?Methods?and?Applications，ed.Innis，Gelland，Shinsky?and?White，Academic?Press，Inc.(1990)。

LCR, similar PCR use the number of many temperature cyclings with the DNA amplification target sequence.Yet LCR does not use independent Nucleotide to be used for template to be extended.On the contrary, LCR depends on the excessive oligonucleotide that is complementary to two chains of target region.After the double-stranded template DNA sex change, the LCR method is since the connection of two Oligonucleolide primers, and described primer is complementary to the adjacent area of one of target chain.The oligonucleotide that is complementary to arbitrary chain can link to each other.Connect and second denaturing step after, primary template strand and two products that reconnect as the templates that connect in addition so that the index amplification of target sequence to be provided.This method has been described in detail among the Genomics, and among the 4:560-569 (1989), it is incorporated herein by reference herein.The same with other amplification system of development, they also can obtain to use in the present invention.

Hybridization medium of the present invention and process are particularly suitable for step analysis.Medium can commercially available acquisition or in the laboratory pre-preparation to comprise the component of hybridizing all needs.For example, analyze damping fluid and the washing composition that medium can comprise chaotropic agent (for example guanidine thiocyanate) expectation at sandwich, comprise the capture probe that is attached to such as the intercalator pseudonucleus thuja acid on the solid support of microballon, and detect the nucleic acid that can also comprise intercalator pseudonucleus thuja acid, yet described nucleic acid needn't necessarily comprise intercalator pseudonucleus thuja acid.Then when analyzing, this medium need only with the sample mix that comprises target nucleic acid.In case hybridize, can wash hybridization complex that is attached to solid support and the degree of determining hybridization.

In order to detect or separated nucleic acid sequence, the sandwich analysis be can commercially available acquisition useful hybridization analysis.This analysis and utilization Covalent Immobilization on solid support " catching " nucleic acid and solution in " signal " nucleic acid of mark.Sample will provide target nucleic acid." catch " hybridization of nucleic acid and " signal " nucleic acid probe and target nucleic acid and form " sandwich " hybridization complex.For effectively, signal nucleic acid is designed to it can not be with capture nucleic acid hybridization but hybridize with the target nucleic acid of different positions except that capture probe.Effectively hybridization can by use intercalator pseudonucleus thuja acid described herein to reduce nucleic acid or nucleic acid analog and the suitable site intercalator pseudonucleus thuja acid that is relative to each other or with suitable site RNA or RNA quasi-molecule the affinity of relevant intercalator pseudonucleus thuja acid, and the affinity that increases simultaneously nucleic acid more described herein or nucleic acid analog is guaranteed.

Oligonucleotide analogs of the present invention comprises that the intercalator oligonucleotide has comprised signalling system (fluorophor) thus.Therefore when oligonucleotide analogs of the present invention used as capture probe, intercalator can be used for detecting the degree of hybridization.Capture probe also can be used for being used in combination with " signal " nucleic acid of mark, and wherein signal nucleic acid can be or can not be oligonucleotide analogs of the present invention and the specificity and/or the susceptibility that can increase analysis like this.

In fact arbitrary solid surface can comprise film, glass, metal and plastics as the upholder of hybridization analysis.Two class solid surface are normally useful, just:

A) film, polystyrene bead, nylon, tetrafluoroethylene, polystyrene/rubber bead, rubber bead or arbitrary solid support with activation silane, carboxylate, sulphonate, phosphoric acid ester or similar activating group are suitable for use as nucleic acid or oligonucleotide and can fix solid surface subgrade on it.

B) the porous-film with preactivate surface that can commercially available acquisition (for example, PallImmunodyne Immunoaffinity Membrane, Pall BioSupport Division, EastHills, N.Y., perhaps Immobilon Affinity membranes from Millipore, Bedford, Mass.) and its can be used for the oligonucleotide of fixed trapped.Also microballon be can use, magnetic bead, alumina bead, polystyrene, tetrafluoroethylene, nylon, silica or rubber bead comprised.

Comprise that the capture probe of intercalator pseudonucleus thuja acid can be attached to the surface of container, described surface adapts with the pcr amplification technology that adopts usually.

The signal nucleic acid that is applicable to the sequence of catching or is used for hybridization analysis can obtain from the genomic complete sequence of organism, and perhaps its part is from mRNA, perhaps from the cDNA that obtains by reverse transcription mRNA.The method that obtains nucleotide sequence from the sequence of this acquisition is well known by persons skilled in the art (referring to the Current Protocols in MolecularBiology of Ausubel etc., pub.John Wiley ﹠amp; Sons (1998), and people's such as Sambrook MolecularCloning, A Laboratory Manual, Cold Spring Habor Laboratory Press, 1989).

In addition, many sequence libraries disclosed and commercially available acquisition all are utilizable and can be used for obtaining correlated series.

The definite of hybridization degree can be undertaken by arbitrary method well known in the art.If there is not detectable hybridization, the hybridization degree just is 0.Oligonucleotide analogs of the present invention comprises the intercalator that can be used to directly detect hybridization.Oligonucleotide of the present invention in addition or oligonucleotide analogs can combine with one or more detectable marks.Complementary nucleic acid or signal nucleic acid can be by being generally used for detecting a kind of mark that carries out in the several method of hybridizing the polynucleotide existence.The detection method of normal use is to use antibody fluorophore or the chemoluminescence agent bonded part with mark.Yet probe also can be used ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³³P or ³²P mark and detecting by radioautography subsequently.Radioisotopic selection depends on and makes the synthetic research preference of simplifying, and changes stability and selects the isotopic transformation period.Other mark comprises the antibody of the specific binding members that can be used as tagged ligand.Use the selection of oligonucleotide analogs of the present invention to have or none or a plurality of other labeled nucleotide depend on sensitivity specificity and the individual's preference that needs.Selective marker depends on sensitivity, simplifies with the probe bonded, and stability needs and suitable plant and instrument.

Can imagine that detection probes is the situation of DNA or RNA.This probe of selection that depends on mark is mark in many ways.Radioactive probe is made by the commercially available radioisotopic Nucleotide that comprises expectation usually.The radioactive nuleus thuja acid can for example be inserted in the probe by several method, such as the nick translation by double-chain probe; Have the strand M13 plasmid of specificity insert under the condition that radioactivity dNTP exists by copy, described insert is the crin promise fragment of archaeal dna polymerase; Under the condition that radioactivity dNTP exists by using reversed transcriptive enzyme to transcribe cDNA from the RNA template; Under the condition that radioactivity rNTP exists, come the RNA of the carrier of self-contained SP6 promotor or T7 promotor by use SP6 or T7 rna polymerase transcribe; The normal PCR that comprises hot dNTPs; By using the 3 ' tailing of terminal enzyme (DNA) with radioactivity nucleotide pair probe; Perhaps pass through to use [ ³²P]-ATP and polynucleotide kinase be to 5 ' terminal phosphateization.

Usually by indirect mode mark nonradioactive probe.Usually, one or more ligand molecular is covalently bound on the probe.Part combines with anti-ligand molecular then, and described anti-ligand molecular can be detected or be covalently bound to signalling system naturally, such as detectable enzyme, on fluorescent chemicals or the chemiluminescence compound.Part and anti-part can extensively change.Wherein part has natural anti-part, biological example element, thyroxine and hydrocortisone, and it can be used for being connected with the naturally occurring anti-part of mark.Perhaps, arbitrary haptens or antigen compound can be used for making up with antibody.

The oligonucleotide analogs that the present invention mentions also can directly be attached to the compound that produces signal in some embodiments, for example by combining with enzyme or fluorophor.The target enzyme that serves as a mark mainly is lytic enzyme, especially Phosphoric acid esterase, and esterase and Glycosylase or oxydo-reductase be peroxidase especially.Fluorescent chemicals comprises fluorescein and derivative thereof, rhodamine and derivative thereof, dansyl, 7 Hydroxycoumarins or the like.Chemiluminescence compound comprises fluorescein, AMPPD ([3-(2 '-spiroamantane)-4-methoxyl group-4-(3 '-phosphinylidyne oxygen)-phenyl-1, and 2-dioxetane]) and 2,3-dihydroxyl two naphthalenediones of mixing, for example, luminol.

The amount that is present in the label probe in hybridization medium or the extraction solution can change within a large range.Usually, the unnecessary greatly probe above the stoichiometric calculation target nucleic acid can be used for increasing the probe amount that is attached to target DNA.Usually can quicken hybridization speed by reaction vessel being immersed commercially available supersound process groove with ultrasonication.

The temperature of the specific cross solution that is suitable for using and under the time cycle hybridization after, with capture probe: (sodium-chlor is for example introduced and contained usually in the washing lotion that is provided in the similar reagents in the hybridization solution to the upholder of target DNA hybridization complex absorption, damping fluid, organic solvent and washing composition).These reagent can be and the hybridization medium similar concentrations, but usually be lower concentration when needing stricter wash conditions.Upholder remain in the washing lotion time cycle can from minute to several hrs or more than.Hybridization or washing medium can be strict.After the suitable strictness washing, can detect correct hybridization complex now according to the characteristic of mark.

Probe can directly combine with mark.For example when being labeled as fluorescence, with first irradiating and detecting of hybridization complex matrix bonded probe with special wavelength light.This light of absorption of sample sends the light of different wave length then, and the light of described different wave length obtains (PhysicalBiochemistry, Freifelder, D., W.H.Freeman ﹠amp by wave-detector; Co.1982), 537-542 page or leaf).When being labeled as radioactivity, sample is exposed to X line film or fluorescence mapping etc.If mark is an enzyme, sample detects in the suitable substrates of enzyme by incubation.The signal that produces can be colored throw out, colour or fluorescence soluble material, the perhaps photon that produces by noclilucence or chemoluminescence.

When mark is enzyme, preferably use and produce the analysis that colored throw out shows positive reading, for example enzyme can be selected from horseradish peroxidase, alkaline phosphatase, calf intestine alkaline phosphatase, glucose oxidase and beta-galactosidase enzymes.For example, alkaline phosphatase is with dephosphorylate indolol phosphoric acid ester, so it will participate in reduction reaction conversion tetrazolium salts to highly colored and insoluble formazan.

The mixture that the detection of hybridization complex can need to produce signal is attached to target duplex and probe polynucleotide or nucleic acid.Usually, this combination is similar to part bonded probe by part and anti-ligand interaction and anti-part is the same with the combination of signal carries out.The combination that produces the signal mixture also can be quickened easily by being exposed to ultrasonic energy.

Mark also can the indirect detection hybridization complex.For example, when mark is haptens or antigen, can be by using the antibody test sample.In these systems, by with fluorescence or the enzyme molecule is connected with antibody or sometimes produce signal by connecting radio-labeling.(Tijssen, P. " application of enzyme immunoassay and theory (Practice and Theory of EnzymeImmunoassays) ", Laboratory techniques in Biochemistry and MolecularBiology, Burdon, R.H., van Knippenberg, P.H., Eds., Elsevier (1985), 9-20 page or leaf).

In the context of the invention, term " mark " is meant that itself or conduct detect a part of detectable group in the series.The example of the funtion part of reporter group is vitamin H, digoxigenin, fluorophor (group that can absorption of electromagnetic radiation, for example light of a certain wavelength or X-ray, and its energy of re-emissioning subsequently and absorbing as long wavelength radiation more; The example of enumerating is dansyl (5-dimethylamino)-1-naphthalene sulfonyl base); DOXYL (N-oxygen-4; 4-Er Jia Ji oxazolidine); PROXYL (N--oxyl-2; 2; 5; the 5-tetramethylpyrrolidi-e), TEMPO (N-oxygen-2,2; 6; 6-four-methyl piperidine), dinitrophenyl, acridine; tonka bean camphor; Cys3 and Cys5 (Biological Detection System, the trade mark of Inc), erytrosine; coumaric acid; umbelliferone; texas Red; rhodamine; tetramethylrhodamin; Rox; 7-nitro benzo-2-oxa---1-diazole (NBD); pyrene; fluorescein; europium; ruthenium; samarium; and other rare earth metals); radioisotopic mark, chemiluminescent labeling (mark that can detect by optical radiation during the chemical reaction), (free radical is (for example for rotary label; the organic nitroxide that replaces) or other with can be by biomolecules bonded paramagnetic probe (for example, the Cu of electron spin resonance spectroscopy detection ²+, Mg ²+), enzyme (such as peroxidase, alkaline phosphatase, beta-galactosidase enzymes and monose oxydase), antigen, antibody, haptens (can still can not begin the group of immunne response with antibodies separately, such as peptide and steroid hormone) carrier system of membrane permeability such as fatty acid residue, steroid part cholesteryl), the somatomedin (PDGF) of the group of the folic acid peptide of vitamin A, vitamins D, vitamin-E, specific receptors, mediation endocytosis, Urogastron (EGF), bradykinin and thrombocyte origin.The example of particularly important is pyrene, anthracene, anthraquinone, vitamin H, fluorescein, texas Red, rhodamine dinitrophenyl, digoxigenin, ruthenium, europium, Cy5, Cy3 etc.

Detect the method for sequence-specific RNA

The present invention also relates to detect the method for sequence specific DNA in the mixture that comprises nucleic acid and/or nucleic acid analog (target DNA), described method comprises the steps:

A) provide nucleic acid mixture; And

B) provide one or more different oligonucleotide or oligonucleotide analogs, wherein the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and corresponding DNA (DNA crossbred) is significantly higher than the melting temperature(Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and corresponding RNA (RNA crossbred), and wherein said oligonucleotide or oligonucleotide analogs are complementary to described sequence-specific RNA basically; And

D) described mixture and described oligonucleotide or oligonucleotide analogs are incubated under the condition of hybridization, thereby seal arbitrary sequence specific DNA; And

F) detect described detectable label; And

Detect described sequence-specific RNA thus.

An advantage of described method is the RNA that can sequence-specific detects in the mixture that comprises similar sequence RNA and DNA.Therefore, the background signal from DNA significantly reduces.Particularly importantly when existing a high proportion of and RNA target to have the DNA of identical or similar sequence (except DNA comprises dT and be U in RNA).

In one embodiment of the invention, the said target mrna or the rRNA that can come by the genetic transcription that in situ hybridization comes the cell (for example bacterium or yeast) of self-contained high copy number plasmid to have detects.

Preferably mixture comprises DNA and RNA.More preferably, mixture does not comprise other nucleic acid or the nucleic acid analog except that DNA and RNA.

Detection comprises that the employed preferred oligonucleotide of method of sequence-specific RNA in the mixture of nucleic acid and/or nucleic acid analog or oligonucleotide analogs are to be selected from the oligonucleotide that comprises at least one aforesaid intercalator pseudonucleus thuja acid or the oligonucleotide or the oligonucleotide analogs of oligonucleotide analogs.

This oligonucleotide or oligonucleotide analogs for example can comprise 3 to 100, such as 3 to 30, such as 3 to 20, such as 3 to 10, such as 10 to 15, for example 15 to 20, such as 20 to 30, for example 30 to 100 Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid.Preferably, described oligonucleotide or oligonucleotide analogs are included in Nucleotide and/or nucleotide analog and/or the intercalator pseudonucleus thuja acid between 10 to 50.

Yet mixture also can be possible for slightly or perhaps experienced the cell extract of purge process.For example mixture can be to have carried out gel electrophoresis and the trace cell extract to the film.

Detectable mark can be directly or indirect detection.For example detectable mark can be enzyme, fluorophor, chromophoric group, radio isotope or heavy metal or any mark of here describing.This foreign labeling can be by the antigenic determinant of the antibody specific identification that comprises mark or by the chemical group of the combination that comprises mark to concrete identification.

Therefore the method that detects RNA comprises by DNA being hybridized to blocking-up of oligonucleotide of the present invention or oligonucleotide analogs and DNA specificity bonded step.Label probe can only not done with the homology complementary RNA with DNA mutually subsequently.This method can be carried out by multiple diverse ways.Any method of for example above-mentioned detection DNA can be used to detect RNA with DNA specificity bonded step by increasing blocking-up.

The method that suppresses RNases and DNases

Because comprise that the oligonucleotide analogs of intercalator pseudonucleus thuja acid of the present invention is more stable to the effect of nuclease than normal nucleic acid, can be suitable for combination and suppress nuclease so comprise one or more concrete oligonucleotide analogs sequences of intercalator pseudonucleus thuja acid.

Therefore, an object of the present invention is to provide the oligonucleotide analogs that comprises RNA or RNA class nuclease and intercalator pseudonucleus thuja acid of the present invention, wherein said oligonucleotide analogs can be under the same conditions suppresses the effect of described RNases for more time in conjunction with one or more various RNases and than the identical oligonucleotide analogs of same amount, and its unique difference with described identical oligonucleotide analogs is to lack described intercalator pseudonucleus thuja acid.

Similarly, an object of the present invention is to provide the oligonucleotide analogs that comprises DNA or DNA class nuclease and intercalator pseudonucleus thuja acid of the present invention, wherein said oligonucleotide analogs can be under the same conditions suppresses the effect of described DNases for more time in conjunction with one or more various DNases and than the identical oligonucleotide analogs of same amount, and its unique difference with described identical oligonucleotide analogs is to lack described intercalator pseudonucleus thuja acid.

Therefore an aspect of of the present present invention relates to and suppresses RNAses and/or the active method of DNAses, described method comprises and increases an at least a intercalator pseudonucleus thuja acid that comprises at least here definition to RNAses and/or DNAses, makes oligonucleotide and/or oligonucleotide analogs in conjunction with RNAse or DNAse and suppress the activity of described RNAse or DNAse thus.

Similarly, an object of the present invention is to provide nuclear base that comprises arbitrary type or the oligonucleotide analogs of examining base analogue and intercalator pseudonucleus thuja acid of the present invention, wherein said oligonucleotide analogs can be under the same conditions suppresses the effect of described nuclease for more time in conjunction with one or more different nucleases and than the identical oligonucleotide analogs of same amount, and unique difference of its described identical oligonucleotide analogs is to lack described intercalator pseudonucleus thuja acid.

Use

Comprise that intercalator pseudonucleus thuja acid ground oligonucleotide and/or the oligonucleotide analogs that define can be used for using usually in arbitrary application of oligonucleotide here.

Especially comprise that the oligonucleotide of at least one intercalator pseudonucleus thuja acid and/or oligonucleotide analogs can be used for following application and/or as following product:

Comprise the linear oligonucleotide and/or the oligonucleotide analogs that embed the pseudonucleus thuja acid

Generalized hybridization probe

The probe of Watson-Crick base pairing

The probe that triplex (Hoogstein base pairing) forms

The probe that chain is invaded

Double-stranded probe of invading

Catch/probe of the random dna of the several sequences of purifying

Catch/probe of purifying sequence specific DNA

By removing the probe of sequence specific DNA purifying RNA

By repetition DNA element (sequence-specific) thus remove the probe of genomic dna purifying RNA

Thereby remove the probe of genomic dna purifying RNA by sequence at random

The probe in blocking dna background hybridization site when carrying out RNA hybridization

Detect the probe of nucleic acid by IPN fluorescence sequence-specific

Detect the probe of nucleic acid by the traditional method sequence-specific

Probe by IPN fluorescence and traditional method binding sequence specific nucleic acid detecting

The primer of Standard PC R (high affinity, highly sensitive reduce unspecific hybridization, reduce primer dimer, reduce false negative, reduce false positive)

The primer of single-basic extension

The primer of the amplification (isothermal duplication, lift-over cyclic amplification) that non-PCR relies on

Primer (w./wo. separates the sequence of amplification from primer) by fluoroscopic examination

Primer by traditional technique in measuring

Combine the primer that detects with traditional method by fluorescence

Be used to increase and the detection primer of hybridization array and detection subsequently

Utilize the SNP detection probes of fluorescence

Utilize two adjacent probe of the SNP detection probes of fluorescence

Utilize single SNP detection probes of melting temperature(Tm)

Utilize two adjacent probe of the SNP detection probes of melting temperature(Tm)

The oligonucleotide analogs array

Gene disruption (transcribing blocking-up)

The oligonucleotide or the oligonucleotide analogs that comprise an intercalator pseudonucleus thuja acid are used to suppress RNases and DNases

Form the oligonucleotide or the oligonucleotide analogs of the intercalator pseudonucleus thuja acid modification of liposome class preparation

The a pair of oligonucleotide analogs that embeds the pseudonucleus thuja acid that comprises preferably all has the pseudonucleus of embedding thuja acid

Use traditional fluorophore/quencher to detect sequence-specific and detect nucleic acid

Use the fluoroscopic examination sequence-specific to detect nucleic acid

Oligonucleotide that nuclease is stable or oligonucleotide analogs

The high affinity bonded duplex structure of oligonucleotide that nuclease is stable or oligonucleotide analogs and Decoy target

Being used for double-stranded duplex of invading sends

Gene disruption

Form the oligonucleotide or the oligonucleotide analogs duplex of the intercalator pseudonucleus thuja acid modification of liposome class preparation

One has the hair clip nucleotide analog of IPNs

The broadest hair clip probe that is used to hybridize

Be used for the hair clip probe that chain is invaded

Be used for double-stranded hair clip probe of invading

Utilize the fluoroscopic examination sequence-specific to detect the hair clip probe of nucleic acid

Use the traditional method sequence-specific to detect the hair clip probe of nucleic acid

Use fluorescence to detect the hair clip probe of nucleic acid in conjunction with the traditional method sequence-specific

The hair clip primer of Standard PC R (high affinity, highly sensitive reduce non-specific hybridization, reduce primer dimer, reduce false negative, reduce false positive)

Hair clip primer (w./wo. separates the sequence of amplification from primer) by fluoroscopic examination

Hair clip primer by traditional technique in measuring

Be used for increasing and hair clip primer that the detection mode of hybridization array and detection is subsequently used

Single SNP by fluoroscopic examination hair clip probe

Two SNP by fluoroscopic examination hair clip probe

Comprise that the oligonucleotide of an intercalator pseudonucleus thuja acid or the array of oligonucleotide are used for above-mentioned various objectives.

Liposome class preparation

A pair of oligonucleotide and/or oligonucleotide analogs

Duplex is sent

Suppress RNA bonded molecule and gene disruption

Antisense (RNA)

Antisense combines with gene disruption

Combination with these aspects.

Embodiment

The following examples have been explained the selected embodiment of the present invention and should not be construed as limitation of the present invention.

The abbreviation that the following example is used:

ODN: oligodeoxyribonucleotide

INA: corresponding to the insertion nucleic acid of intercalator pseudonucleus thuja acid

Embodiment 1

The preparation of intercalator pseudonucleus thuja acid

1-pyrene methyl alcohol can commercially availablely obtain, but it also can provide 1-(chloromethyl) pyrene of 98% productive rate to prepare easily by the Vilsmeier-Haack formylation succeeded by reducing and carry out ethanol conversion with thionyl chloride subsequently with sodium borohydride by pyrene.

By (S)-(+)-2,2-dimethyl-1,3-dioxalane-4-methyl alcohol and 1-(chloromethyl) pyrene prepares acyclic amidite 5 (accompanying drawing 1) with 52% overall yield.Utilize KOH to carry out alkylated reaction; and utilize 80% aqueous acetic acid to produce glycol 3 (accompanying drawing 1) and finished the synthetic of 5 (accompanying drawings 1); its with dimethoxy trityl chloride (DMT-CI) protection and with 2-cyanoethyl N; N; N ', N '-tetra isopropyl phosphoric acid diamines carry out the purpose compound 5 (accompanying drawing 1) that end reaction provides 72% productive rate.When with 2-cyanoethyl N, N-di-isopropyl chlorine phosphorus amidite during as the phosphitylating reactant productive rate in later reaction stage reduce to 14% from 72%.The composite diagram of acyclic amidite 5 is shown in the accompanying drawing 1.

1-pyrenyl formaldehyde

Cooling N-formyl radical-methylphenylamine (68.0g on ice-bath; 41.4mL; 503mmol) and the mixture of o-dichlorobenzene (75mL) and add phosphoroxychloride (68g; 440mmol) exceeding two hours makes temperature be no more than 25 ℃.Add a spot of pyrene powder (50g in 30 minutes; 247mmol) and with condenser reaction cartridge mixture and 90-95 ℃ heating 2 hours.The bolarious compound of elimination is also with benzene (50mL.) washing behind the cool to room temperature.Change over to then also to stir in the water (250mL) and spend the night.

(3 * 50mL) washings of elimination xanchromatic aldehyde and water.Carry out recrystallization 3 times with 75% ethanol.Output: 30.0g (52.7%).

1-pyrenyl methyl alcohol

In 10 minutes inherent rare gas elementes with 1-pyrenyl formaldehyde (10.0g; 43.4mmol) be dissolved in anhydrous THF (50mL) and add minor N aBH ₄(0.82g; 22mmol).This product of water (350mL) crystallization that stirred reaction mixture spends the night and stirs by impouring under the room temperature.Leach product, water (4 * 25mL) washing and drying under reduced pressure.Carry out recrystallization with ethyl acetate.Output: 8.54g (84.7%).

1-(chloromethyl)-pyrene

With 1-pyrenyl methyl alcohol (6.40g; 27.6mmol) be dissolved in pyridine (3.3mL; 41.3mmol) and CH ₂Cl ₂In the mixture (100mL) and mixture be cooled to 0 ℃.In 15 minutes, slowly add SOCl ₂(3.0mL; 41.3mmol) and allowable temperature slowly rise to room temperature.Stirring is spent the night.In the water (200mL) that the mixture impouring is stirred and add CH ₂Cl ₂(100mL).Stirred the mixture 30 minutes.Use 5%NaH respectively ₂CO ₃(2 * 75mL) and salt solution (2 * 75mL) washing organic phases are with dried over sodium sulfate and carry out concentrating under reduced pressure.Carry out recrystallize with toluene/sherwood oil.Output 6.75g (97.8%).

(S)-(+)-2,2-dimethyl-1.3-dioxalane-4-methyl alcohol

With powdery KOH (25g) and 1-(chloromethyl)-pyrene (6.0g; 23.9mmol) join (S)-(+)-2,2-dimethyl-1,3-dioxalane-4-methyl alcohol (2.6g; 19.7mmol) dry toluene (250mL) solution in.The mixture 16h that under the Dean-Stark condition, refluxes, cool to room temperature and add entry (150mL) then.(3 * 100mL) washing organic phases, sal epsom and dried over sodium sulfate mixture and concentrating under reduced pressure are a kind of viscous crude to water.Silica gel chromatography separates (CH ₂Cl ₂) the pure compound of 6.1g (90%) is provided.

(R)-3-(1-pyrenyl methoxyl group)-propane-1.2-diphenol

(S)-(+)-2,2-dimethyl-1,3-dioxalane-4-methyl alcohol (6.1g; 17.6mmol) be added into acetate and water (100mL; 4: 1) mixture in and room temperature under stir 19h.Concentrating under reduced pressure.Produce the oil of certain output.

(S)-1-(4,4 '-the dimethoxytrityl methoxyl group)-3-pyrenyl methoxyl group-2-propyl alcohol

With (R)-3-(1-pyrenyl methoxyl group)-propane-1,2-diphenol (760mg; 2.48mmol) be dissolved in anhydrous pyridine (20mL) and add dimethoxy trityl chloride thing (920mg; 2.72mmol).Reaction mixture is stirred 24h and concentrating under reduced pressure.Produce a kind of white foam by silica gel chromatography purifying (ethyl acetate/hexanaphthene/triethylamine 49: 49: 2).Output 1.20g (79.5%).

(S)-phosphoramidite of 1-(4,4 '-dimethoxytrityl methoxyl group)-3-pyrenyl methoxyl group-2-propyl alcohol

(S)-1-(4,4 '-the dimethoxytrityl methoxyl group)-3-pyrenyl methoxyl group-2-propyl alcohol (458mg; 753 μ mol), 2-cyanoethyl-N, N, N ', N '-tetra isopropyl phosphine (453mg; 429 μ l; 1.51mmol) and di-isopropyl ammonium tetrazolium (193mg; 1.13mmol) sneaked into anhydrous CH ₂Cl ₂Stirred 6 days (7ml) and in oxide gas.By silica gel chromatography (ethyl acetate/hexanaphthene/triethylamine 49: 49: 2) purifying and drying under reduced pressure.Output 438mg (72%) is white foam.

Embodiment 2

3-(1-pyrenyl methoxyl group)-propane-1, the selectable synthetic method of 2-glycol

Schema 1:3-(1-pyrenyl methoxyl group)-propane-1, the selectable synthetic method of 2-glycol

1-pyrenyl methyl alcohol (232mg; 1.0mmol) be dissolved in heat toluene (2mL over Na).Add CsF (7mg; 0,046mmol) also when adding 3-chloro-1,2-propionic aldehyde glycol (170mg; 1.53mmol) time at room temperature stirred about 1 hour.80 ℃ of 2h that stir the mixture, cool to room temperature and filtering mixt are separated to precipitated product.With cold toluene washing (2 * 1mL).Output 220mg (72%).

Embodiment 31-O-4,4 '-dimetoxytrityl-4-O-(9-antracenylmethyl)-1,2, the 2-O phosphoramidite of 4-trihydroxybutane synthetic

Schema 2:1-O-4,4 '-dimetoxytrityl-4-O-(9-antracenylmethyl)-1,2, the synthetic synoptic diagram of the 2-O phosphoramidite of 4-trihydroxybutane

9-anthracene methyl chloride (II)

With 9-anthracene methyl chloride (0.81g; 3.89mmol; I) be dissolved in anhydrous pyridine (467 μ l; 5.83mmol) and anhydrous CH ₂Cl ₂Under 0 ℃ of stirring, dropwise add SOClO ₂(423 μ L; 5.83mmol), the 24h that stirs the mixture, during temperature be allowed in 2 hours, be elevated to room temperature.In the water that reactant is stirred by impouring (60mL) and add additional C H ₂Cl ₂(40mL).Use 5%NaHCO respectively ₃(100mL) solution, salt solution (100mL) and water (100mL) washing organic phase.Pass through Na ₂SO ₄Carry out drying and vacuum concentration.Output 665mg (75%).

1,2-D--isopropylidene-4-(9-anthryl methyl)-1,2,4-trihydroxybutane (III)

With 9-anthracene methyl chloride (628mg; 277mmol) be dissolved in anhydrous toluene (25mL over Na) and 2-[(S)-2 ', 2 ' dimethyl-1 ', 3 '-dioxalan-4 ' yl]-ethanol (506mg; 3.5mmol) and the KOH that adds 3 little spoons.Be connected Dean-Stark instrument and under refluxad stir and spend the night of mixture.Reaction mixture is by cool to room temperature slowly and use H ₂O (4 * 25mL) washings.

Pass through Na ₂SO ₄Dry also vacuum concentration.

4-O-(9-anthryl methyl)-1.2.4-trihydroxybutane (IV)

Stirred reaction mixture 24h under the AcOH (50mL) of adding 80% and the room temperature in dry compound.Spent the night vacuum concentration and by of short duration, the fast silica gel chromatogram method is carried out purifying and (at first used CH of mixture ₂Cl ₂Wash-out impurity, and use the CH of 5% methyl alcohol then ₂Cl ₂The eluant solution product).By two step productive rates is 56.3%.

1-O-(4,4 '-dimethoxytrityl)-4-O-(9-anthryl methyl)-1,2,4-trihydroxybutane (V)

Glycol (425mg; 1.40mmol) and DMT-CI is sneaked into, and (5mL) also at room temperature stirred 36 hours in the anhydrous pyridine.The vacuum concentration reaction mixture and by the silica gel chromatography purifying (EtOAc: ^CC ₆H ₁₂: N (Et) ₃63: 35: 2).After separating little yellow foam, carry out coevaporation with ether (5mL overNa).Output 630mg (74%).

1,2-O-(4,4 '-dimethoxytrityl)-4-O-(9-anthryl methyl)-1,2, the inferior phosphinylidyne of 4-trihydroxybutane Amine (VI)

The anthracene compound of DMT protection is dissolved in anhydrous CH ₂Cl ₂(7mL) di-isopropyl ammonium tetrazolide (252mg; 1.5mmol) and add 2-cyanoethyl N, N, N ', N '-tetra isopropyl phosphine.Stirred reaction mixture 20h under the room temperature.Vacuum concentration and by silica gel chromatography (EtOAc: ^CC ₆H ₁₂: N (Et) ₃24: 74: 2) purifying.Produce a kind of weak yellow foam (67%) with ether (5mL over Na) coevaporation.

Embodiment 4

(S)-1-(4,4 '-the dimethoxytrityl methoxyl group)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone)-2-butanols (V)

Schema 3:_ (S)-1-(4,4 '-dimethoxytrityl methoxyl group)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone)-synthetic (V) of 2-butanols

7,9-dimethyl-pyrido [3 ', 2 ': 4.51 thieno-s [3,2-d] pyrimidines-4 (1H)-ketone (I)

According to diplomatic method preparation 7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone ^1,2,3

3-N-((S)-2 ", 2 "-dimethyl-1 ", 3 "-dioxalane-4 "-ethyl group) 7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone (II)

¹Schimdt，U.&Kubitzek，H.，Chem.Ber.，93，1559(1960)

²Hassan, K.M. etc., Phosporous, Sulfur, Silicon Relat.Elem., 47,181 (1990)

³Gewald，K.&Jnsch，H.J.，Prakt.Chemie313-320(1976)

With 7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone (1.16g; 5.0mmol) be suspended in the dry DMF (20mL) and add NaH (0.2g; 5.0mmol, the suspension of 60%meneral oil).The 2h that stirs the mixture emits up to all H2 and stops.Add (S)-2 then, 2-dimethyl-1,3-dioxalane-4-ethanoyl-O-is right-toluenesulphonic acids fat (0.78g; 5.1mmol) and at 80 ℃ of 24h that stir the mixture.The evaporated in vacuo mixture is with dry toluene vacuum coevaporation and separate (5%EtOAc in CHCl3) purifying resistates by silica gel column chromatography and obtain a kind of colourless product.Output 0.81g; 45%.

(S)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone-butane- 1,2-glycol (III)

In 80% acetate (20mL), stir under the room temperature 3-N-((S)-2 ", 2 "-dimethyl-1 ", 3 "-dioxalane-4 "-ethyl group)-7,9-dimethyl-pyrido [3 ', 2:4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone (0.75g; 2.1mmol) 24h.Obtain product by vacuum concn with the EtOH coevaporation.Separate (5% methyl alcohol in CHCl3) purifying by silica gel column chromatography and obtain colourless product.

Output 0.5g (75%).

(S)-1-(4,4 '-the dimethoxytrityl methoxyl group)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] Thieno-[3,2-d] pyrimidines-4 (1H)-ketone)-2-butanols (IV)

With (S)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone-normal-butyl-1,2-diphenol (0.6g; 1.9mmol)) be dissolved in the anhydrous pyridine (5mL) and add DMT-Cl (0.71g; 2.1mmol).Stirred overnight at room temperature.Vacuum concentration and with dry toluene (3 * 10mL) coevaporations.Produce a kind of white foam by silica gel chromatography chromatography (EtOAc: CC6H12: N (Et) 349: 49: 2) purifying resistates.Output 0.77g (65%).

(S)-1-(4,4 '-(4,4 '-the dimethoxytrityl methoxyl group)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] Thieno-[3,2-d] pyrimidines-4 (1H)-ketone)-phosphoramidite (V) of 2-butanols

In nitrogen with (S)-1-(4,4 '-(4,4 '-dimethoxytrityl methoxyl group)-4-(7,9-dimethyl-pyrido [3 ', 2 ': 4,5] thieno-[3,2-d] pyrimidines-4 (1H)-ketone)-2-butanols (310mg; 0.5mmol) be dissolved in anhydrous methylene chloride (10mL).Add diisopropylaminoethyl tetrazolium (0.11g; 0.67mmol) dropwise add 2-cyanoethyl-N then, N, N ', N '-tetra isopropyl phosphoro group diamide fat (0.3g; 1.0mmol), be reflected in the nitrogen to stir and spend the night, vacuum concentration and by the silica gel chromatography chromatography (EtOAc: ^cC ₆H ₁₂: N (Et) ₃49: 45: 12) purifying produces a kind of white foam.Output 345mg (84%).

Embodiment 5

Duplex with unsettled end.

Be the accumulation ability of research nucleoside analog 5 (accompanying drawing 1), it is added to two difference self complementary strands (end of 5 '-XCGCGCG and 5 '-XTCGCGCGA).

Utilize the GeneAssembler Special software of 1.53 versions to carry out the synthetic of ODN by Pharmacia LKB Gene Assembler Special.Amidite is dissolved in anhydrous acetonitrile with pyrene, and the widow-nucleotide chain that is prepared into the solution of 0.1M and insertion growth under the same conditions carries out normal Nucleotide coupling (2min, coupling).The coupling efficiency of the Nucleotide of modifying is greater than 99%.Synthesize ODNs with DMT, and carry out purifying by Waters Delta Prep 3000 HPLC that on Hamilton PRP-1 post, have Waters 600E controller and Waters 484 detectors.Buffer A: 950ml.0.1M NH ₄ ⁺CO ₃ ^-+ 50ml MeCN pH=9.0; Buffer B: 250ml NH ₄ ⁺CO ₃ ^-+ 750ml MeCN pH=9.0.Gradient: 5min.100%A, 40min internal linear gradient arrives linear gradient and the interior 100%A (product peak ≈ 37min.) of 29min of 100%A to 100%B in the 5min.100%B, 1min.ODNs is at 925 μ l H ₂O+75 μ lCH ₃Carry out the DMT surrender among the COOH and protect and utilize identical post once more by HPLC, buffering system and gradient are carried out purifying (product peak ≈ 26min.).For removing possible salt, ODNs is dissolved in the water of 1ml and vacuum concentration 3 times again.

All oligonucleotide are carried out the MALDI-TOF analysis and are confirmed by the Voyager EliteBiospectrometry Research Station with PerSeptive Biosystems.Utilize PETEMPrev.5.1 software and PECSS software package ver.4.3 to carry out the analysis of transition state by PerkinElmer UVNIS spectrometer λ 2 with PTP-6 programmable temperature device.At 1M NaCl, 10mM sodium phosphate, pH7.0, the melting temperature(Tm) of mensuration self complementary sequence in every kind of DNA chain of 1.5 μ M.At 150mM NaCl, the 10mM sodium phosphate, 1mM EDTA pH7.0 measures all other ODNs in each chain of 1.5 μ M.All melting temperature(Tm)s have ± 0.5 ℃ uncertainty.

Carrying out the Amber field of force in the MacroModel 6.0 and 7.0 of water as solvent calculates and minimizes by Conjugant Gradient method.The initial oligonucleotide sequence of pyrene that is used to calculate insertion is available from the Brookhavens albumen database, and modified in MacroModel before beginning to minimize.Lam and Au-Yeung by NMR explained with this research in the structure of used identical self complementary sequence.Their structure prolongs and is used to the reckoning of structure with pyrene amidite at 5 of each chain ' end.Other sequence is the long terminal repetition zone of the HIV-1 of 13-monomeric unit high conservative.G-7 is replaced by pyrene amidite and calculates the C-Nucleotide that has and do not have intersection.Pyrene is placed in the amphiploid inside from initiator.All keys are moved and rotation freely.

Modify with unmodified, the melting temperature(Tm) of self complementary DNA is presented in the accompanying drawing 33.Adding pyrene amidite as 5 of unsettled end ' end as depend on those that 5 stabilizations at the bonded duplex of nucleic acid chains 5 ' end are similar to Guckian etc. the people found based on the base stabilized DNA duplex of band underscore 19.2 ℃-21.8 ℃ (each modifies 8.6 ℃-10.9 ℃), its 5 ' end at self complementary ODNs has inserted pyrene nucleosides (oligodeoxynucleotide).Stable can the explanation, its predict by utilizing " MacroModel " to calculate be with the base pair of being with underscore in the interactional pyrene part of two nucleosides (accompanying drawing 2).

Table 1. has the melting temperature(Tm) of 5 ' self complementary sequence of modifying.

T _m(℃)????ΔT(℃)

5′C-G-C-G-C-G

41.0

3′G-C-G-C-G-C

5′T-C-G-C-G-C-G-A

46.9

3′A-G-C-G-C-G-C-T

5′X-C-G-C-G-C-G

62.8??????21.8

3′G-C-G-C-G-C-X

5′X-T-C-G-C-G-C-G-A

64.1??????17.2

3′A-G-C-G-C-G-C-T-X

Embodiment 6

Depend on the stabilization introduction of end-position insertion pseudonucleus thuja acid of intercalator-joint length

Shown that in this embodiment depending on joint length to increase affinity by inserting the pseudonucleus thuja acid to 5 of oligonucleotide ' end.Two examples that further shown the insertion pseudonucleus thuja acid that can compare stabilization in addition.

Material and method

Oligonucleotide:

Probe I: 3 '-CGA ACT CX

Probe I I:3 '-CGA ACT CD

Probe I II:3 '-CGA ACT CY

Ref：3’-CGA?ACT?C

Target: 5 '-GCTTGAG

Below shown the amidites that is used to prepare above-mentioned oligonucleotide:

Utilize target chain and the probe chain of 1.5 μ M in containing the 2mL buffered soln of following material, to carry out all hybrid experiments:

140mM?NaCl

10mM?Na ₂HPO ₄·2H ₂O

1mM?EDTA

pH＝7.0

Anneal by target chain and probe being mixed 3min in above-mentioned damping fluid, afterwards cool to room temperature at leisure at 95 ℃.By heating the solution in the quartz cuvette at leisure, measure the melting temperature(Tm) of probe-target mixture that light absorption ratio obtains hybridizing simultaneously.Record all melting temperature(Tm)s in these examples by revision test and have uncertain ± 1.0 ℃.

Result and discussion

The result who tests that unwinds is presented in the table 5:

Title	Melting temperature(Tm) (℃)	????ΔTm(℃)
Title	Melting temperature(Tm) (℃)	????ΔTm(℃)	Hybridize with target
????Ref	????22.8	????----	Hybridize with target
????Ref	????22.8	????----	Probe I	????28.4	????5.6
Probe I I	????34.4	????11.6	Probe I	????28.4	????5.6
Probe I I	????34.4	????11.6	Probe I II	????33.8	????11.0

The difference of melting temperature(Tm) depends on the short circuit head of probe I between probe I and the II.Therefore joint and intercalator is best in conjunction with length, is important for increasing greatly of affinity between oligonucleotide that obtains the modification of insertion pseudonucleus thuja acid and the target DNA sequence.Probe I I and III have the affinity for its target sequence much at one, although the insertion portion in two probes is very different.These show that the pseudonucleus thuja acid of insertion is that a class depends on that required feature should be incorporated into the compound in oligonucleotide or the oligonucleotide analogs, and it should design according to strict rule more or less.

Embodiment 7

The oligonucleotide that synthetic oligonucleotide on general upholder-acquisition 3 ' intercalator pseudonucleus thuja acid is modified

In a preferred embodiment of the invention, oligonucleotide or oligonucleotide analogs are included in the insertion pseudonucleus thuja acid of one or two end.Shown that in these embodiments the oligonucleotide or the oligonucleotide analogs that have an intercalator pseudonucleus thuja acid at 3 ' end can utilize universal support to synthesize.It shows that further containing self complementary oligonucleotide that inserts the pseudonucleus thuja acid at 3 ' end has formed very heat-staple hybrid.

Material and method

Self complementary probe of following two types is synthesized.Utilize general upholder synthesis type A and utilize standard nucleotides coupling column and the synthetic B of method:

Type A)

And B)

Two different insertion pseudonucleus thuja acids that are used for type A (I and II), and II also is used for type B.Reference sequences without any insertion pseudonucleus thuja acid (III) is synthesized.Therefore X represents:

Or III)

After synthetic, be used in 32%NH ₄2%LiCl in the OH solution handles oligonucleotide analogs with the blocking group of removing heterocyclic amine and the oligonucleotide of the general upholder of cracking.Contain the evaluation that the oligonucleotide that inserts the pseudonucleus thuja acid is experimentized by MALDI-TOF and obtained expection.

All cross experiments are to utilize two targets of 1.5 μ M in 1mL buffered soln and probe chain to carry out, and buffered soln contains:

140mM?NaCl

10mM

1mM?EDTA

By 95 ℃ in above-mentioned damping fluid hybrid target chain and probe they were annealed in 3 minutes.Thereafter cool to room temperature at leisure.By heating the solution in the quartz cuvette at leisure, measure the melting temperature(Tm) of probe-target mixture that light absorption ratio obtains hybridizing simultaneously.Record all melting temperature(Tm)s in these examples by revision test and have uncertain ± 1.0 ℃.

Result and discussion

???? The X type	A?????????B
???? The X type	A?????????B	? ???? I? ????II ????III	57.3℃????------ ? ? 59.2℃????62.8℃ 41.0℃????41.0℃

Insert the pseudonucleus thuja acid and increased affinity arbitrary terminal embedding of oligonucleotide as can be known from the above table for complementary target nucleic acid.Same its shown that also the pseudonucleus thuja acid that inserts can be inserted into 3 ' end of oligonucleotide widow or nucleotide analog by the general basic chemistry that utilizes standard.

Embodiment 8

With 1-O-(pyrenyl methyl) glycerol Nucleotide substituted nucleotide

As described in embodiment 5, carry out the synthetic of ODN.As preparation phosphoramidite 5 (accompanying drawing 1) as described in the embodiment 1.

Wherein G Nucleotide passes through flexibility, the joint ethylene glycol and 1 of no base, and the substituted UV melting temperature(Tm) of ammediol is measured (table 2), and not unexpected the demonstration, the stability of comparing duplex with unmodified, full complementary sequence has reduced.Synthesize the cyanoethyl N of the DMT protection of required ethylene glycol, NN, N-di-isopropyl phosphorus-amidites and 1, ammediol by standard method.The glycol that 1-O-(pyrenyl methyl) glycerol Nucleotide replaces no base in same position has increased by 16.4 ℃-18.0 ℃ of the melting temperature(Tm)s of DNA/DNA duplex, the both sides that show pyrene and duplex be coaxial to because the stabilization of each modification has surpassed the effect (table 1) that the pyrene unit is placed an end of duplex.The reckoning of carrying out according to " MacroModel " shows, the nucleoside base that is inserted into and inserts the 5 ' side in site and 3 ' side when the pyrene unit only produces double-helical than small deformation (accompanying drawing 3) when having in the interactional duplex.The stabilization that duplex is undertaken by the coaxial accumulation of pyrene part can not compensate fully and depend on that hydrogen bond passes through partly to replace with pyrene the loss in conjunction with affinity of the minimizing quantity of G, and the duplex of modification is compared less stablized 8.6 ℃ with the complete complementary duplex of unmodified.The DNA/RNA duplex is found has identical trend, although these have lower melting temperature(Tm) than corresponding D NA/DNA duplex usually.The stabilization of pyrene part is for only 8.2 ℃ of DNA/RNA duplexs when comparing with ethylene glycol, and the stable of DNA/DNA duplex turns to 16.4 ℃.The insertion of pyrene causes having between the ssDNA of 9.0 ℃ of differences of melting temperature(Tm) of corresponding duplex and the ssRNA and has produced the difference that increases.

One of them Nucleotide of table 2 is no base, flexible joint, pyrene unit (5), a DNA/DNA disappearance or complementary G and DNA/RNA duplex.

U-U/??????U-U/

5′A-G-C-T-T-G-C-C-T-T-G-A-G

3′T-C-G-A-A-C-X-G-A-A-C-T-C

DNA RNA difference

Clauses and subclauses T _m(℃) T _m(℃) Δ T _{M, DNA-RNA}(℃)

1????? ???26.0????25.8??????0.2

2?????

????????27.6????26.8??????0.8

3??????X＝5??????????????????????44.0????35.0??????9.0

4??????X＝-(12-mer)??????????????35.2????29.6??????4.6

5??????X＝G(13-mer)??????????????52.6????47.2??????5.4

Embodiment 9

1-O-(pyrenyl methyl) glycerol is as ridge

Usually import ridge and in duplex, reduce melting temperature(Tm).Equally also here observe by (table 3), if the pyrene unit is embedded into as ridge, the melting temperature(Tm) of DNA duplex rises 3 ℃.These observationss with people such as Ossipov are consistent, promptly find must import a large amount of instability that ridge prevents duplex as the non-Watson-Crick of importing during in conjunction with intercalator.Compare with flexible ethylene glycol joint, 11.2 ℃ of partially stabilized duplexs of pyrene have shown that pyrene partly is inserted in the duplex.

When inserting a pyrene modification as ridge, the difference of the melting temperature(Tm) between the DNA/RNA duplex that DNA/DNA duplex that pyrene is modified and pyrene are modified is increased to 12.6 ℃.Big 7.4 ℃ and in these diversity ratio unmodified duplexs than big more of the difference between the duplex that contains natural nucleosides or flexible ethylene glycol ridge.This means that pyrene partly is optionally and only can stabilized DNA/DNA duplex and unstable DNA/RNA duplex.For one duplex of back, it exists duplex partly to have identical melting temperature(Tm) with the glycerol joint rather than with pyrene, shows that pyrene is not inserted in this chain.

The structure of the DNA/DNA structure that pyrene is modified calculates that (accompanying drawing 4) shows that the pyrene unit only produces less distortion in duplex, and joint is introduced competent flexibility and made pyrene partly and have the distance of 3.4 between the nucleic acid base of same chain in main chain.The nucleic acid base of chain has the distance shorter slightly than 3.4 of optimum between pyrene part and nucleic acid base relatively.

For further studying difference and stabilization phenomenon, preparation has the ODNs that two pyrene amidite insert, and understand whether the unitary effect of pyrene is addition, and it really.Result's (table 3) shows, relies on the distance between two insets and their the adjacent base pair, compares with natural DNA duplex, and (each modification is stablized 6.7 ℃) is possible up to 13.4 ℃ to stablize the pyrene DNA/DNA duplex that twice inserts.How many unstable of the DNA/RNA duplex that pyrene is modified is addition too, and therefore when inserting two pyrenes modifications, the difference of the melting temperature(Tm) between the DNA/RNA duplex that DNA/DNA duplex that pyrene is modified and pyrene are modified is up to 25.8 ℃.For stabilization and difference, when inserting respectively, obtained best result by 4 base pairs.When two insets of pyrene amidites are adjacent one another are in ODN, to compare with the DNA/DNA duplex of unmodified, melting temperature(Tm) has reduced by 5.2 ℃ and compare with the duplex of single modification and to have reduced by 8.2 ℃.It should be noted that when comparing a base pair in the DNA/DNA duplex between two insets is enough to improve DNA/DNA stabilization and DNA/RNA difference with only having the duplex of an inset.

Table 3 has the different hybridization that replace in the melting temperature(Tm) of the oligonucleotide of DNA or RNA.

Embodiment 10

Five different pseudonucleus thuja acids that insert as ridge inset with avidity that the complementary DNA target is increased

Shown the general introduction of in the middle of the DNA oligonucleotide, inserting five different insertion pseudonucleus thuja acids below.Described embedding pseudonucleus thuja acid is as the ridge inset when hybridizing with target.The oligonucleotide that all insertion pseudonucleus thuja acids that here show are modified is compared with the oligonucleotide of unmodified has the avidity that the complementary DNA target is increased:

Target dna strand 5 ' A-G-C-T-T-G-C-T-T-G-A-G T _{M, DNA}

3′T-C-G-A-A-C-G-A-A-C-T-C??????????????????????????????????47.4℃

???????????50.4℃

???????50.6℃

????????49.4℃

Shown below joint and intercalator in conjunction with length, clearly all examples have much at one intercalator and joint in conjunction with length (9.9 ± 1.3 ).

Our the pseudonucleus thuja acid that can conclude insertion is the compound in conjunction with the simple rule of length requirement that a big group meets intercalator and joint thus.

Embodiment 11

To DNA than high affinity-to RNA than low-affinity

Shown in this embodiment when the target nucleic acid sequence with respect to probe is DNA, by import inserting the pseudonucleus thuja acid and increased in the probe melting temperature(Tm) of hybrid-no matter probe is DNA or RNA (referring to following table 4).No matter probe is DNA or RNA, reduced in addition for the affinity of RNA target.Therefore, intercalator pseudonucleus thuja acid can introduce that oligonucleotide or oligonucleotide analogs produce avidity that oligonucleotide or oligonucleotide analogs increase DNA and to RNA and class RNA compound as LNA, 2 '-avidity that the O-methyl RNA reduces.

U-U/???????????U-U/

5 ' A-G-C-T-T-G-X ₁-C-X ₂-T-T-G-A-G chain 1

3 ' T-C-G-A-A-C-X ₃-G-X ₄-A-A-C-T-C chain 2

/U????????????????????????????/U

Chain 1 chain 2 Tm (℃)

Type X X type X ₃X ₄

₁?? ₂

DNA duplex and crossbred

DNA??????????????-???-????DNA??????-????-????48.4

DNA??????????????5???5????DNA??????-????-????51.4

DNA??????????????-???-????DNA??????5????5????48.4

RNA duplex and crossbred

RNA??????????????-???-????RNA??????-????-????57.8

RNA??????????????5???5????RNA??????-????-????46.8

RNA??????????????-???-????RNA??????5????5????47.2

The DNA/RNA crossbred

DNA??????????????-???-????RNA??????-????-????42.2

DNA??????5??5????RNA????-???-????34.2

DNA??????-??-????RNA????5???5????45.2

The different situation of table 4:3 kind.At the top: owing to have intercalator pseudonucleus thuja acid in the crossbred, the DNA duplex avidity compare with the duplex that does not have chain to contain intercalator pseudonucleus thuja acid be increase or do not change.In the centre: owing to have intercalator pseudonucleus thuja acid in the crossbred, it is unsettled that the RNA duplex is compared with the duplex that does not have chain to contain intercalator pseudonucleus thuja acid.In the bottom: if shown that the RNA chain contains intercalator pseudonucleus thuja acid then crossbred between DNA and the RNA chain is stabilized, shown in addition when being incorporated into the DNA chain, be lowered for the avidity of RNA.5=is incorporated in the chain according to above-mentioned method from the amidite 5 of embodiment 1.

By the different relative positions of intercalator pseudonucleus thuja acid, higher avidity was possible during acquisition comparison DNA was shown in and shows.This embodiment is presented in the table among the embodiment 9.

Embodiment 12

The cross hybridization that reduces

Cross hybridization between two corresponding oligonucleotide comprises that at least one has the intercalator pseudonucleus thuja acid of the reduction avidity that depends on intercalator pseudonucleus thuja acid relative position.If shown intercalator in the following table each other just relatively, how many melting temperature(Tm)s reduced.

U-U/???????????U-U/

5 ' A-G-C-T-T-G-X ₁-C-X ₂-T-T-G-A-G chain 1

3 ' T-C-G-A-A-C-X ₃-G-X ₄-A-A-C-T-C chain 2

/U?????????????????????????????/U

Chain 1 chain 2 Tm (℃)

DNA duplex and crossbred

DNA?????????????????-?????-???DNA????-????-??????48.4

DNA?????????????????5?????5???DNA????-????-??????51.4

DNA????5????5????DNA????5????5????42.6

RNA duplex and crossbred

RNA????-????-????RNA????-????-????57.8

RNA????5????5????RNA????-????-????46.8

RNA????5????5????RNA????5????5????45.4

DNA and RNA crossbred

DNA????-????-????RNA????-????-????42.2

DNA????-????-????RNA????5????5????45.2

DNA????5????5????RNA????5????5????37.8

The different situation of table 5:3 kind.At the top: owing to have intercalator pseudonucleus thuja acid in the crossbred, DNA duplex is with not have chain to contain the DNA duplex of intercalator pseudonucleus thuja acid stabilized.When the hybridization of oligonucleotide or oligonucleotide analogs, if intercalator pseudonucleus thuja acid is made them closely face mutually each other toward each other, then with when only a chain comprises intercalator pseudonucleus thuja acid, compare melting temperature(Tm) and be lowered.In the centre: owing to have intercalator pseudonucleus thuja acid in the crossbred, it is unsettled that the RNA duplex is compared with the duplex that does not have chain to contain intercalator pseudonucleus thuja acid.In the bottom: if shown that intercalator pseudonucleus thuja acid is included in the RNA chain then the crossbred between DNA and the RNA chain is stabilized.Further shown when the hybridization of oligonucleotide or oligonucleotide analogs, if intercalator pseudonucleus thuja acid makes them closely face mutually each other toward each other, then with when only a chain comprises intercalator pseudonucleus thuja acid, compare melting temperature(Tm) and be lowered.To be added in the chain from the 5=amidite 5 of embodiment 1 according to above-mentioned method.

Embodiment 13

Fluorescence

Be incorporated into the reduction of the fluorescence of the single pyrene modifying DNA chain on the complementary strand, show that pyrene is inserted in the duplex.The oligonucleotide that the twice pyrene inserts produces same result for all different ODNs.Ratio and ssRNA hybridization more remarkable (accompanying drawing 5 and 6) when the DNA that modifies is hybridized with ssDNA show that less pyrene is inserted in the DNA/RNA duplex.These have supported the conclusion of pyrolysis chain experiment, are promptly protruded by glycerol in the DNA/RNA duplex and pyrene almost to have identical melting temperature(Tm) and draw and lack in the DNA/RNA duplex that pyrene is inserted into protrusion (table 3).

By separated two pyrene parts of Nucleotide only, because the formation of pyrene residue excimer, at one the 3rd peak of generation, 480nm place.When this class had the DNA of two pyrene insets and complementary dna chain hybridization, this band almost disappeared.This shows that inserting a complete base pair has prevented that two pyrenes from partly entering the physical distance that excimer forms required about 3.4nm.When double inset DNA and complementary RNA hybridization, two pyrene parts still can interact, because found a real excimer band.

Embodiment 14

Contain the oligonucleotide of insertion pseudonucleus thuja acid or the 3-exonuclease stability of oligonucleotide analogs

Material and method

DNA is with reference to I, the snake venom phosphorus diesterase degradation time process of the mixture of INA oligonucleotide II and I and II.With 1.2U snake venom phosphorus diesterase [the following buffered soln of 30 μ L: 5mM Tris-HCI; PH=7.5; 50% glycerine (v/v)] under the room temperature degraded at 2mL damping fluid (0.1MTris-HCI; PH=8.6; 0.1M NaCl; 14mM MgCl ₂) in the solution (each chain in the test of the mixture of 1.5 μ M) of 1.5 μ M of all chains.

DNA?oligo：3’-TGT?CGA?GGG?CGT?CGA

INA?oligo：5’-YAC?AGC?YTC?CCY?GCA?GCY?T

The result

Utilize snake venom phosphorus diesterase (SVPDE) to estimate INA (containing the nucleic acid that inserts the pseudonucleus thuja acid) oligonucleotide and compare accompanying drawing 16 for the stability of the circumscribed degraded of 3 '-nucleic acid and with the stability of normal DNA.It has shown with reference to DNA oligonucleotide I, has fully been degraded by SVPDE within 15 minutes.Opposite INA only only demonstrated little hyperchromic effect and does not observe significant hyperchromic effect thereafter within first 15 minutes.These experiments show 3 of INA oligonucleotide '-terminal DNA Nucleotide degraded by SVPDE, but it stops and ability is further degraded when enzyme touches the first insertion pseudonucleus thuja acid.

Its medium chain I and II be the blended experiment in the SVPDE test, has produced hybridization.Observe degraded slowly with independent comparing with reference to the DNA chain.Observing the DNA chain after 60 minutes is almost degraded completely.These results show that crossbred compares by slower degraded with single stranded DNA.

Embodiment 15

The hair clip shape oligonucleotide that embeds the pseudonucleus thuja acid that contains that is used for detection of nucleic acids

Introduce

Shown in this embodiment and contained the detection how the hair clip shape oligonucleotide (probe 1) that embeds the pseudonucleus thuja acid is used to nucleic acid.It has shown that further it is possible utilizing these principles to detect low target nucleic acid to 5nM solution (1pmol is in 200 μ L).It has shown that also adding cetyl trimethylammonium bromide (HTMAB) can strengthen the signal sensitivity in the material that concentration relies on.

Material and method

Shown the sequence that contains the detection probes of inserting the pseudonucleus thuja acid below.The Nucleotide that relates to hair clip formation is added UnderscoreAnd be shown as black matrix in conjunction with the relevant Nucleotide of target:

Probe 1:5 '- CAT CCG YAYAAG CTT CAA TCG GAT GGT TCTTCG

The secondary structure that in accompanying drawing 17, has shown hair clip.It is done the hydrogen bond of going up base and is shown as a little.

The sequence that has shown used target in these experiments below.Participate in detection probes bonded Nucleotide and be shown as black matrix:

Target: 3 '-ATA GTA TTT ATT CGA AGT TAG CCT ACC AAG AAGCCT TTT TTG

All hybrid experiments carry out in containing the damping fluid of following substances:

140mM?NaCl

10mM?Na ₂HPO ₄·2H20

1mM?EDTA

pH＝7.0

Used tensio-active agent is HTMAB in the experiment.

In accompanying drawing 18, shown the accompanying drawing of explanation when probe is incorporated into its target sequence.It has shown and has no longer been separated by a complete base pair from two pyrene parts inserting the pseudonucleus thuja acid when probe and target hybridization.This makes them more freely interact, and produces higher excimer fluorescence:

In following table, shown Experimental design:

The hole	??1	??2	??3	??4	??5	??6	??7	??8
The hole	??1	??2	??3	??4	??5	??6	??7	??8	??a	??H ₂O	Damping fluid	Probe I 100pmol	Probe I 10pmol	Probe I 1pmol	Probe I 100pmol+target 100pmol	Probe I 10pmol+target 10pmol	Probe I 1pmol+ target 1pmol
??b	??H ₂O+ tensio-active agent 10 ^-6	Damping fluid+tensio-active agent 10 ^-6	Probe I 100pmol+tensio-active agent 10 ^-6	Probe I 10pmol+tensio-active agent 10 ^-6	Probe I 1pmol+ tensio-active agent 10 ^-6	Probe I 100pmol+target 100pmol+tensio-active agent 10 ^-6	Probe I 10pmol+target 10pmol+tensio-active agent 10 ^-6	Probe I 1pmol+ target 1pmol+ tensio-active agent 10 ^-6	??a	??H ₂O	Damping fluid	Probe I 100pmol	Probe I 10pmol	Probe I 1pmol	Probe I 100pmol+target 100pmol	Probe I 10pmol+target 10pmol	Probe I 1pmol+ target 1pmol
??b	??H ₂O+ tensio-active agent 10 ^-6	Damping fluid+tensio-active agent 10 ^-6	Probe I 100pmol+tensio-active agent 10 ^-6	Probe I 10pmol+tensio-active agent 10 ^-6	Probe I 1pmol+ tensio-active agent 10 ^-6	Probe I 100pmol+target 100pmol+tensio-active agent 10 ^-6	Probe I 10pmol+target 10pmol+tensio-active agent 10 ^-6	Probe I 1pmol+ target 1pmol+ tensio-active agent 10 ^-6	??c	??H ₂O+ tensio-active agent 10 ^-5	Damping fluid+tensio-active agent 10 ^-5	Probe I 100pmol+tensio-active agent 10 ^-5	Probe I 10pmol+tensio-active agent 10 ^-5	Probe I 1pmol+ tensio-active agent 10 ^-5	Probe I 100pmol+target 100pmol+tensio-active agent 10 ^-5	Probe I 10pmol+target 10pmol+tensio-active agent 10 ^-5	Probe I 1pmol+ target 1pmol+ tensio-active agent 10 ^-5
??d	??H ₂O+ tensio-active agent 10 ^-4	Damping fluid+tensio-active agent 10 ^-4	Probe I 100pmol+tensio-active agent 10 ^-4	Probe I 10pmol+tensio-active agent 10 ^-4	Probe I 1pmol+ tensio-active agent 10 ^-4	Probe I 100pmol+target 100pmol+tensio-active agent 10 ^-4	Probe I 10pmol+target 10pmol+tensio-active agent 10 ^-4	Probe I 1pmol+ target 1pmol+ tensio-active agent 10 ^-4	??c	??H ₂O+ tensio-active agent 10 ^-5	Damping fluid+tensio-active agent 10 ^-5	Probe I 100pmol+tensio-active agent 10 ^-5	Probe I 10pmol+tensio-active agent 10 ^-5	Probe I 1pmol+ tensio-active agent 10 ^-5	Probe I 100pmol+target 100pmol+tensio-active agent 10 ^-5	Probe I 10pmol+target 10pmol+tensio-active agent 10 ^-5	Probe I 1pmol+ target 1pmol+ tensio-active agent 10 ^-5

95 ℃ of down all probes and target annealing 2.5 minutes in 200 μ L damping fluids of Eppendorf pipe respectively, and slowly be cooled to room temperature then and change 96-hole black microwell plate from NUNC over to.By having the Wallac Victor of following specification ², 1420 Multilabel gaugers are measured fluorescence:

Emission filter: F340

Excitation filter: 500-10F

Minute: 0.1s, the 4.0mm place from the microwell plate bottom.

CW-lamp energy: 50054, constant voltage control.

Result and discussion

Below shown the result who measures:

The hole	???1	???2	???3	???4	???5	????6	???7	???8
The hole	???1	???2	???3	???4	???5	????6	???7	???8	??a	??4299	??3471	??4927	??3204	??2639	??20643	??4988	??3841
??b	??5709	??3619	??9684	??4563	??3644	??18611	??3971	??4308	??a	??4299	??3471	??4927	??3204	??2639	??20643	??4988	??3841
??b	??5709	??3619	??9684	??4563	??3644	??18611	??3971	??4308	??c	??4021	??3119	??8429	??1879	??3456	??13833	??5337	??2202
??d	??2236	??3194	??120959	??12749	??1898	??223956	??21684	??4783	??c	??4021	??3119	??8429	??1879	??3456	??13833	??5337	??2202

Relatively a3 and a6 as seen, probe I is hybridized have been increased fluorescence greatly in the target chain and has proved the principle of utilizing hair clip shape oligonucleotide detection nucleotide sequence.If background correction level value (a2) from measure, then observing probe I has increased almost 12 times by the fluorescence with its target sequence hybridization.

Detect as can be seen by a4 relatively and a7 and a5 and a8 that to hang down to the existence of being reduced to the target nucleic acid of 1pmol by 10pmol be possible.

Equally also the display surface promoting agent has increased fluorescence level.Add the HTMAB tensio-active agent and increased fluorescence in some cases, increase the sensitivity of detection thus up to 100 times above 100 times (post 6).

These results can compare as the fact of the primer in the direct extension of template with probe I, make the detection of oligonucleotide or oligonucleotide analogs at for example nucleic acid, be used for labeling nucleic acid, being used for connection of extension picture and PCR and real-time quantitative PCR becomes a kind of very useful instrument.

Embodiment 16

Few Jie He ﹠amp on the SAL-chip; The regulation and control of INA-signal.

Method

The chip preparation

Oligonucleotide, 50 μ M, point is dissolved in 400mM sodium carbonate buffer, pH9.

Immediately chip is inserted in the wet cell, 37 ℃ one hour.

By using 1%NH ₄The oligonucleotide combination was carried out in the OH washing in 5 minutes on chip surface.

With the deionized water wash 2 * 2 minutes of heating not.

600rpm centrifugal chip 5 minutes is to remove the excessive water on surface.

Scanning chip, or 4 ℃ of stored under refrigeration.

Oligonucleotide bonded SYBR Green II contrasts dyeing

Deionized water with not heating washed 2 * 2 minutes at about 90 ℃.600rpm is centrifugal 5 minutes subsequently.

The dilution SYBR Green of 10 000x II is added on the chip, adds a cover and at room temperature incubation 2-3 minute.

The water washing that utilization is not heated 1 minute, and the 600rpm centrifugal chip made its drying in 5 minutes.

Utilize Alexa 488 filterset scanning chip at once.

The result

Chip section is divided from OUH, has the HUMAC of amino-joint oligonucleotide with SYBR Green II dyeing.

Referring to accompanying drawing 19

Point size: 100 μ m.Center-width between centers: 175 μ m

The evaluation widow-and the combination (referring to accompanying drawing 20) of INA-on Asper SAL-chip.

INA oligonucleotide with signal-modification and amino joint is inferred to be incorporated into the SAL chip as normal amino joint oligonucleotide.(referring to SYBR Green Yes:1,2,3,4 and 7).

When blueness or blue-greenish colour wave filter are used for our ArrayWorx scanning device, all signal-INAs fluoresce (SYBR Green, No:1,2,3,4).

The position of signal-modification with few-5 '-end compares and looks not remarkable.Depend on no matter be to greatest extent away from 5 '-terminal (1) or the most close 5 '-do not observe evident difference between the strength of signal of the modification of terminal (4).

Short 10-mere INAs does not have joint, and obviously debond is in chip-they lack joint (referring to 5).The intensity of signal with when only with damping fluid oligonucleotide point sample of no use, be identical.(referring to SYBR Green Yes, 5﹠amp; 6)

The background that shows the variation of identical chips different piece may be because inadequate washing, the calibration of scanning device, or the variation of SAL coating causes.

Viewed trend: containing the characteristic of INA modification time point usually when oligonucleotide, also is shape and signal-homogeneity, looks it is (to compare 1 and 7, the right of bottom) preferably.

Embodiment 17

DNA-DNA, INA-DNA and the hybridization characteristic of INA-INA crossbred in different pH values

Introduce

Shown DNA-DNA in this embodiment, INA-DNA and the hybridization avidity of INA-INA crossbred in different pH values.

Material and method

Crossbred:

Crossbred I 5 '-CTCAACCAA GCT

3’-GAG?TTG?GTT?CGA

Crossbred II 5 '-CTC AAC YCA AGC T

3’-GAG?TTG?GTT?CGA

Crossbred III 5 '-CTCAACCAA GCT

3’-GAG?TTG?YGT?TCG?A

Crossbred IV 5 '-CTC AAC YCA AGC T

3’-GAG?TTG?YGT?TCG?A

All hybrid experiments are to carry out in 1mL contains the damping fluid of following substances with the target of 1.5 μ M and probe chain:

140mM?NaCl

10mM

1mM?EDTA

By 95 ℃ in above-mentioned damping fluid hybrid target chain and probe annealed 3 minutes.Cool to room temperature at leisure afterwards.By heated solution slowly in quartz curette, and measure the melting temperature(Tm) of probe-target crossbred that its light absorption ratio obtains hybridizing simultaneously.Measure all melting temperature(Tm)s among this embodiment by repeated experiments to have ± ℃ uncertainty.

NH with 25% ₄The solution of OH and Glacial acetic acid is adjusted pH.

Result and discussion

Crossbred #	?pH ?4.2??5.0?????6.1?????7.0?????8.0?????9.0????10.0
Crossbred #	?pH ?4.2??5.0?????6.1?????7.0?????8.0?????9.0????10.0	? I? II III IV	?30.6?43.6????47.8????47.2????49.6????49.6???43.3 ? ? ?-????49.7????54.7????54.5????55.7????54.1???51.1 ?-????45.4????51.9????52.5????54.7????52.1???46.4 ?-????36.1????46.0????46.5????48.5????46.5???40.9

Last table has shown the melting temperature(Tm) result of experiment of crossbred-IV in different pH values.Crossbred II and III have higher melting temperature(Tm) than homologous DNA duplex (crossbred 1) and crossbred IV from the pH scope of pH=5 to 10 as can be seen from the table.This shows that when described sequence hybridization, two sequences are when at least one insertion pseudonucleus thuja acid is contained in the position toward each other, the cross hybridization that reduces between the complementary sequence is possible.Equally also as can be seen when the pH=8 left and right sides melting temperature(Tm) of all crossbreds be the highest, therefore preferably some hybridization in some preferred embodiments in pH=8 ± 2.Containing one, to insert the crossbred II and the III of pseudonucleus thuja acid and contain the maximum difference that inserts the melting temperature(Tm) between the crossbred IV of pseudonucleus thuja acids at two relative positions be during at pH=5.0, promptly is respectively 13.6 ℃ and 9.3 ℃.Therefore in a preferred embodiment, containing hybridization between at least one oligonucleotide that inserts the pseudonucleus thuja acid or oligonucleotide analogs and nucleic acid or the nucleic acid analog o'clock carries out in pH=5 ± 1.

Embodiment 18

Preparation is used for the sample of RT-PCR

The method that preparation is used for the sample of target sequence RT-PCR is described in accompanying drawing 7.This method has an advantage, and promptly the false positive signal from DNA is reduced widely.

Cell sample is provided and destroys the cell walls of cell, discharge the DNA and the RNA (accompanying drawing 7A) of cell thus.Subsequently, can hybridize the oligonucleotide that contains intercalator pseudonucleus thuja acid, allow under the condition of hybridizing between oligonucleotide and the DNA with DNA/RNA sample incubation (accompanying drawing 7B) in target sequence.Pass through the RT-PCR method amplification sample (accompanying drawing 7C) of arbitrary standards then.Because the target DNA of sample quilt is by blocking with oligonucleotide hybridization, only RNA can be amplified then.

Selectable, destroy after the cell walls, for example come purifying RNA (accompanying drawing 7D) by standard method arbitrarily by extraction and precipitation.Usually, purified RNA will contain a spot of DNA pollutent.Therefore, can hybridize the oligonucleotide that contains intercalator pseudonucleus thuja acid, allow under the condition of hybridizing between oligonucleotide and the DNA with DNA/RNA sample incubation (accompanying drawing 7E) in target sequence.Then by the sample (accompanying drawing 7F) of standard RT-PCR method amplification arbitrarily.Because the target DNA pollutent of sample quilt is by blocking with oligonucleotide hybridization, only RNA can be amplified then.

Embodiment 19

Preparation is used for the sample of RT-PCR

The method that preparation is used for the sample of target sequence RT-PCR is described in accompanying drawing 8.This method has the advantage that the false positive signal from DNA is reduced widely.

Cell sample is provided and destroys cell walls, released dna and RNA thus.By standard method purifying arbitrarily from the RNA (accompanying drawing 8B) of sample, yet the step that the DNA/RNA sample is carried out subsequently also is possible.

Sample is with the magnetic bead incubation that is connected in the oligonucleotide that contains intercalator pseudonucleus thuja acid (accompanying drawing 8C), and it can be hybridized in target sequence under the condition of hybridizing between permission oligonucleotide and the DNA.Hybridization back sample be filtered separate with sample in target DNA bonded magnetic bead (accompanying drawing 8D).

Prepare sample and be used for RT-PCR (accompanying drawing 8E).Because from the DNA of sample separation, the false-positive danger of RT-PCR that causes owing to the DNA pollutent is reduced the sequence-specific target widely.

Selectable, after the specimen preparation, sample is with the solid support incubation that is connected in the oligonucleotide that contains intercalator pseudonucleus thuja acid (accompanying drawing 8C), and it can be hybridized in target sequence under the condition of hybridizing between permission oligonucleotide and the DNA.Hybridization back solid support separates from sample with the bonded target DNA.Sample can be once more with being connected in the solid support incubation that contains intercalator pseudonucleus thuja acid, separates the sequence specific DNA in the sample of remaining in of trace.After sequence specific DNA hybridization, from sample, remove solid support (accompanying drawing 9C).

Sample prepares for RT-PCR then.

Embodiment 20

The purifying of sequence specific DNA

The purifying of sequence specific DNA is shown in accompanying drawing 9 and 10.

Handle cell sample with GnSCN and discharge nucleic acid thus.Sample is with the magnetic bead incubation that is connected in the oligonucleotide that contains the intercalator oligonucleotide (accompanying drawing 10A), and it can be hybridized in target sequence under the condition of hybridizing between permission oligonucleotide and the DNA.Sample is filtered and washs-bonded nucleic acid non-(accompanying drawing 10B) to remove.Heating and filter magnetic bead discharges purely, is substantially free of the sequence specific DNA (accompanying drawing 10C) of sequence-specific RNA.

Selectable, sample is with the solid support incubation that is connected in the oligonucleotide that contains intercalator pseudonucleus thuja acid (accompanying drawing 11B), and it can be hybridized in target sequence under the condition of hybridizing between permission oligonucleotide and the DNA.The residuum of separation solid support and sample also heats, and it has discharged sequence specific DNA (accompanying drawing 11C).Sequence specific DNA is substantially free of sequence-specific RNA and prepares for diagnosis, prevention PCR or other purpose.

Embodiment 21

The detection of target DNA

The oligonucleotide that contains pyrene pseudonucleus thuja acid is connected to chip.

Design oligonucleotides makes its part can hybridize in the specificity target DNA and makes this oligonucleotide also can the oneself hybridize.When oligonucleotide hybridization in himself the time, 3 pairs of pyrene pseudonucleus thuja acids toward each other, therefore and the melting temperature(Tm) of DNA/ oligonucleotide hybridization body is higher than the melting temperature(Tm) of self crossbred.In addition, oligonucleotide contains two pyrenes (accompanying drawing 12 and accompanying drawing 13A) that only do not carry out can forming when self hybridizes excimer when probe.

The different oligonucleotide of discerning different target DNA s can be added to the different zones of chip.In the present embodiment, 2 different oligonucleotide are connected to a little 1 and point 2.

The crude mixture that contains the dna fragmentation of target DNA can not be added on the chip under the temperature of self-annealing at oligonucleotide.

Behind annealing steps and the washing step, reduce temperature and allow probe self hybridization.Detect the amount of the existence and the target DNA hybridization of target DNA by the formation of excimer.Method is shown in accompanying drawing 12.

Selectable, but design oligonucleotides makes it contain a fluorophor and a quencher, and wherein the fluorophor signal can be only by quencher cancellation (accompanying drawing 13B) when oligonucleotide oneself hybridization.

Also can utilize two oligonucleotide that respectively contain 3 pyrene pseudonucleus thuja acids respect to one another when oligonucleotide hybridization.Oligonucleotide also respectively contains a fluorophor and a quencher, and it is placed and makes that the fluorophor signal only can be by quencher cancellation (accompanying drawing 13C) when oligonucleotide hybridization.

Embodiment 22

From the exciplex fluorescence that inserts the pseudonucleus thuja acid

Introduce

Two exciplex fluorescence missiles that embed between the pseudonucleus thuja acid have been shown in this embodiment.

Material, method and result

Utilize synthetic 3 of standard method to contain two different oligonucleotide that insert the pseudonucleus thuja acid:

Sequence 1:5 '-CTCAAYGDCAAGCT

Sequence 2:5 '-CTCAAGYDCAAGCT

Sequence 3:5 '-CTCAAGYXCAAGCT

Behind the HPLC purifying, will contain the oligonucleotide that inserts the pseudonucleus thuja acid and be dissolved in the damping fluid that contains following substances:

140mM?NaCl

10mM

1mM?EDTA

pH＝7.0

And all fluorescence experiments are all carried out in this damping fluid.Excite at the 343nm place by PerkinElmer MPF-3 spectrophotometer with xenon 150 supply of power.

From accompanying drawing 21 and accompanying drawing 22 as can be seen, the monomer fluorescence ratio of the exciplex when inserting pseudonucleus thuja acid adjacent (accompanying drawing 21) is higher than (accompanying drawing 22) when being right after nearest neighbor-and can clearly observe the transition of exciplex in both cases.Obtained similar result with amidite X.

As a result, when described pseudonucleus thuja acid is placed on oligonucleotide inside, can be observed the exciplex (referring to accompanying drawing 23) that inserts the pseudonucleus thuja acid between two two insertion pseudonucleus thuja acids adjacent and that be right after nearest neighbor.

Embodiment 23

Carry out PCR with the Oligonucleolide primers that contains intercalator pseudonucleus thuja acid

Plasmid template (1.5mMMgCl in the Standard PC R damping fluid of 25 μ final volume with dilution ₂50mM KCl; 10mM Tris-HCl; 0.1%Triton X-100, the various dNTP of 200DM, the various primers of 5pmol) carry out 35 round-robin grads PCRs (94 ℃, 30 seconds; The gradient annealing temperature, 45 seconds; 72 ℃, 60 seconds).By the separation PCR product of 0.7% sepharose in the 1xTBE damping fluid and by EtBr dyeing developing.Temperature on the accompanying drawing 24 is represented the annealing temperature in each hole.

Design of primers (designing upstream and downstream respectively)

a01??????????5′-????????AAGCTTCAATCGGATGGTTCTTCG

a02??????????5′-???????YAAGCTTCAATCGGATGGTTCTTCG

a03??????????5′-YCYATCCGAAAGCTTCAATCGGATGGTTCTTCG

a05??????5′-CYAYTCCGAAAGCTTCAATCGGATGGTTCTTCG

b01??????????5′-??????CACAAGAGCTGACCCAATGGTTGC

b02??????????5′-????????YCACAAGAGCTGACCCAATGGTTGC

b03??????????5′-YTYGGGTCACACAAGAGCTGACCCAATGGTTGC

b05??????5′-TYGYGGTCACACAAGAGCTGACCCAATGGTTGC

When not hybridizing with target, primer 03 and 05 can form hairpin loop, is illustrated below by 05 primer:

Independent primer TCAA

T??TCGGA?T?GGTTCTTCG-3′

C?AGCCTYAY

GAA????C-5′

Primer 5 '-CYAYTCCGA

AAGCTTCAATCGGATGGTTCTTCG

TTCGAAGTTAGCCTACCAAGAAGC

Target 5 '-TA

CT

Conclusion as from the image viewing of gel to, compare with DNA contrast primer, in linear primer a02/b02, add a single end-position intercalator pseudonucleus thuja acid and improved effective melting temperature(Tm) significantly.

The amplified production of the band of 03 and 05 primer is with height efficient manner initiation beacon design primer PCR.

Embodiment 24

Be incorporated into the INA-oligonucleotide of DNA target

Result and discussion

In conjunction with needs target-specific and spontaneous generation.

Design a series of INA-oligonucleotide and measure them naturally in conjunction with the ability (accompanying drawing 25) of the complementary target dna sequence of 80bp.INA-oligonucleotide (IOs) by 37 ℃ of double-stranded target DNA of incubation and excessive P32-marks in the sodium phosphate buffer that contains 120mM sodium-chlor reacted in 1-3 hour.Analyze by electrophoretic mobility displacement then and phosphorus-video picture evaluation result of mark lOs.

Accompanying drawing 26 has shown that the IOs (IO 1-1, IO 1-2, IO 1-3) of all 3 mensuration is in conjunction with target DNA.Measure relative amount by the volumetric analysis that utilizes ImageQuant software to postpone band in conjunction with IOs.The numeral of accompanying drawing bottom shows the different avidities of IOs for target.IO 1-3 has in conjunction with the advantage of target significantly and therefore is selected for further analysis.

And then determine the evidence of observed binding specificity.At first the IO 1-3 of P32 mark is separately with arbitrary chain incubation of DNA target.Based on the sequence consistency, IO 1-3 specificity is in conjunction with the positive-sense strand (accompanying drawing 27, swimming lane 1-3) of DNA target as expected.When measure increasing the concentration of IO 1-3 of P32 mark then to the combination of double stranded DNA target and and the combination of the 60bp target DNA sequence of nothing to do with compare (comparison swimming lane 4-5 and 7-8).Obviously, the target of not observing the target consistency that needs by IO in conjunction with the combining of the target DNA of sequence-irrelevant.For proving the position of observed delay, a part of target DNA s is by P32 mark and their delay of replicate(determination) (swimming lane 6 and 9).

IO pairing does not suppress spontaneous combination and by inserting target-avidity that unitary different positions changes.

The chain specificity of viewed IOs (being the positive-sense strand of IO 1-3 specificity in conjunction with target DNA, referring to top) shows that the antisense strand of target DNA can at random be attacked by different IO simultaneously.For studying these possibilities, the complementary IOs that IO 1-3 is different with 3 anneals together.The pairing of IOs still makes the IO 1-3 of P32 mark rely on the different avidities of inserting unitary position among the pairing IO even have spontaneously in conjunction with target DNA as shown in Figure 28.Because IO 1-3/IO 5 pairing has produced best target-combination, whether this pairing is selected as further mensuration and studies to match and influenced spontaneous target bonded efficient, IO 1-3 in conjunction with the determined pairing that has and do not have with above-mentioned IO 5.Accompanying drawing 29 shows that pairing does not influence the spontaneous combination of IO 1-3 to target DNA.

Nf promotes IO target combination and IOs pairing

The auxiliary link molecule of the RecA/Rad51 that between DNA target and little RNA-DNA oligonucleotide, forms be in the news in the past (Gamper 2000, and Yoon 2002).IO is not having under the situation of help at an easy rate in conjunction with target and therefore is being hopeful as the bona fide factor that is used for the treatment of the DNA target of purpose.How therefore it be very important for verifying that these are combined in the nuclear environment.Study us and from people's cell culture, extract nuclear in order to carry out this.As shown in Figure 30, under the situation of examining extraction, being delayed to of IO 1-3 reduces and adds 3-5 and doubly (do not calculate and show), and only takes place under the situation that specific target exists.In addition, the bonded degree depends on the amount that nucleoprotein adds increases as such up to adding a certain amount of protein.Then as using the reduction of expecting based on the similar analysis of adding and DNA repair process proteins associated matter is carried out.Usually this process of supposition is relevant with the D-ring formation of target DNA, stays two chain openings and is used for by mating the attack that oligonucleotide carries out.Therefore to have strengthened the IO 1-3 of P32-mark really be significant to the combination (accompanying drawing 31) of DNA target for observing the adding of extracting paired IO ' s by nuclear.

Material and method

Oligonucleotide-synthetic: all oligonucleotide are prepared by standard method

The radio-labeling of oligonucleotide: by with polynueleotide kinase and γ-p ³²ATP incubation together comes the end mark oligonucleotide.Oligonucleotide by Mermaid test kit method purifying mark.

Nuclear extract: prepare the HT-29 extract from the HT-29 colon cancer cell of pre-fusion by the NUN extracting method.The HeLa nuclear extract is available from (a Belgian company).

Electrophoretic mobility is measured: containing 120mM NaCl, reacting in the 20mM sodium phosphate buffer of the pH8.0 of 1mM DTE.Behind 37 ℃ of incubations, the swimming that powers on of 7 among the 1/2xTBE or 10% polyacrylamide glue gel also-80 ℃ is stored or is used directly in reaction solution quick-frozen in liquid N2,300V, and 4 ℃ are carried out 2-4h.

The reaction solution that contains nuclear extract contains the 0.22M urea in addition, the NaCl of total 200mM, 0.22% NP-40,5.52HEPES, 5mM MgCl ₂With 2mM ATP.37 ℃ of incubation 10min of these reaction solutions then add the 10%SDS of 1.175 μ l and 37.5g Proteinase K and other 60 minutes of incubation again.

In order to compare purpose, equate that the independent IOs of CPM is added in the reaction solution.

Video picture: utilize STORM phosporimager and ImageQuant software evaluation EMSA result.

Embodiment 25

LNA+INA: by inserting the nucleic acid hair clip that the nucleic acid monomer preparation is easy to the closure of target

Introduce

Hairpin structure is the common trait of single stranded DNA and RNA sequence.Such secondary structure can produce the target sequence that is difficult at intermolecular generation Watson-Crick base pair ( ⁱ).People need find that new technology alleviates this problem, before those report be utilize nucleotide sequence be adjacent to ( ^Ii) part or use N ⁴-ethyl-2 ' Deoxyribose cytidine is replaced 2 ' natural-Deoxyribose cytidine, its with 2 '-Deoxyribose cytidine form base pair compare with natural base pair stability with reduction ( ^Iii).The oligonucleotide that has developed various modifications last two age people develops the agent of potential gene inhibition, and it has the stability of enhanced pair cell nuclease, the ability of cell membrane infiltration and to the effective hybridization of target RNA/DNA.If the oligonucleotide of modifying has effective hybridization characteristic, they also are hoped to form the secondary structure that can produce a large amount of inaccessible sequences, but our cognition is not attempted overcoming this problem to modified oligonucleotide.

The LNA oligonucleotide is monomer and the 2 '-O that contains a kind of conformation restriction, the oligonucleotide of 4 ' C-methylene bridge (accompanying drawing 1), and when with consistency DNA or RNA hybridization, compare the thermostability that they demonstrate spiral with the duplex of unmodified.Because their hybridization efficiency, they also are supposed to form highly stable hairpin structure.It is also attacked produces the LNA hairpin structure that is easy to target such as LNA, seemingly the most potential antisense material standed in the oligonucleotide of Xiu Shiing.

By insert INAs (inserting nucleic acid, accompanying drawing 1) that the pseudonucleus thuja acid constitutes in the DNA between DNA and the RNA when hybridize with them quilt discern consumingly ( ^Iv).Suitably design than its DNA/DNA pairing produce steady I NA/DNA duplex more, yet find that but it is than the less stable duplex of corresponding RNA/RNA duplex generation when INAs is hybridized with RNA.Show that in this file this characteristic can be used for by inserting the INA monomer to the middle LNA hair clip that is easier to the DNA target that produces of doing of hair clip.

Material and method

Synthesizing of the LNA of DMT protection and INA phosphoramidite

Prepare respectively as previously mentioned LNA and INA phosphoramidite (4a, 8a).

ODN, LNA and INA's is synthetic, the mensuration of purifying and melting temperature(Tm)

By the Expedite 8909 nucleic acid synthesis systems of Applied Biosystems synthetic ODN, LNA and INA.LNA and INA amidite are dissolved in 1: 1 mixture of anhydrous acetonitrile and anhydrous methylene chloride, 0.1M solution, and utilization and normal oligodeoxynucleotide coupled the same terms are inserted in the oligonucleotide chain of growth (coupling in 2 minutes).The coupling efficiency of modified nucleotide is＞99%.Utilize DMT to synthesize ODNs, LNAs and INAs and Waters Delta Prep 3000 HPLC by having Waters 600E controller and Waters 484 detectors on the Hamilton PRP-1 post carry out purifying.The 0.1M NH of buffer A: 950ml ₄HCO ₃With 50ml MeCN, pH9.0; The 0.1M NH of buffer B: 250ml ₄HCO ₃With 750ml MeCN, pH9.0.Gradient: 5 minutes 100%A, 40 minutes linear gradients to 100%B, 5 minutes 100%B, 1 minute to the linear gradient of 100%A and 100%A (the product peak was at 37 minutes) in 29 minutes then.ODNs, LNAs and INAs are at H2O and the 75 μ l CH of 925 μ l ₃Gone protection and by the HPLC purifying, reuse identical post by DMT among the COOH, buffering system and gradient (the product peak was at 26 minutes) desalt for removing, and with ODNs, LNAs and INAs are dissolved in the water of 1ml and vacuum concentration 3 times again.

All ODNs, LNAs and INAs are carried out MALDI-TOF and analyze and prove by the Voyager Elite Biospectrometry Research Station that utilizes PerSeptive Biosystems.Perkin Elmer UVNIS spectrometer λ 2 by having PTP-6 programmable temperature device also utilizes PETEMP rev.5.1 software and PECSS software package v.4.3 to carry out the analysis of transition state.At 120mM NaCl, 10mM, sodium phosphate, 1mM EDTA, pH7.0 measures all ODNs in 3.0 each chain of μ M.Measure all melting temperature(Tm)s by repeated experiments and have uncertain ± 0.5 ℃.

Result and discussion

Duplex

NMR has been used to measure the structure of DNA/LNA duplex and has it is found that only LNA monomer in duplex, the variation of the sugared structure that is enough to induce side joint Nucleotide from the north structure generally B-form DNA/DNA duplex, found to the south structure, the latter generally find in the sugar moieties of A-type RNA/RNA duplex ( ^v).Therefore it is found that research INA monomer P (accompanying drawing 32) this duplex institute might the site system's insertion be very significant.Without any the monomeric oligonucleotide I of LNA be used as with except that 3 '-terminal all possible positions have the reference target that probe 2-12 (accompanying drawing 33) that P inserts is hybridized.In all cases, when comparing with the probe 1 of unmodified, oligonucleotide I has increased the stability of duplex.In fact, probe 3 has produced the duplex that remarkable stable having increased by 10.1 ℃ of pyrolysis chain temperature.This oligonucleotide has INA monomer P in the insertion in AT zone and when comparing with another probe, and this looks usually more desirable.

II observes from oligonucleotide, and when comparing with the probe 1 of unmodified, INA monomer P has increased the stability of duplex in its insertion away from the monomeric duplex of LNA zone.In fact, stability has almost proved the structure that still has the B-type away from the monomeric zone of LNA with the same of oligonucleotide I and these.Have only when P is inserted in the monomeric complementary oligonucleotide of contiguous LNA (accompanying drawing 33,2-5 item), the difference of the hybridization efficiency of two oligonucleotide and their complementary sequence hybridizations can be observed.These may be because the conformational change of the sugar moieties of adjacent nucleotide and these are reflected on the pyrolysis chain decrease of temperature.Main difference is found to be duplex and has the monomeric adjacent insertion of LNA (accompanying drawing 33,2 and 3), and the less important difference of duplex is adjacent insertion with next (accompanying drawing 33,4 and 5).

Having 3 equalitys places the monomeric oligonucleotide III of LNA to be considered in the duplex structure of inducing the A type by the major part of the duplex that forms with its complementary sequence hybridization.These are by comparing the Δ T of oligonucleotide III and oligonucleotide I _mS infers.This conclusion be consistent to having the NMR structure determination that the monomeric similar duplex of 3LNA carries out ( ^v).Similarly stable only find these two in NA monomer P is inserted into oligonucleotide when being adjacent to its corresponding duplex terminal, and so proved terminal Type B duplex (accompanying drawing 33,10 and 11).

To having studies have shown that when the Nucleotide of modifying is induced A type duplex structure in the zone of Type B duplex of oligonucleotide I-III, a kind of general means in A and Type B duplex zone are differentiated in INA monomer P insertion conduct.

Hair clip

By in the experiment of pyrolysis chain, adding the oligonucleotide T of thermosetting hair clip separately ₄-DNA (referring to the sequence key point of accompanying drawing 34) observes because the ssDNA hair clip is opened the considerable change of generation at 37.2 ℃ (accompanying drawing 2A and accompanying drawings 34).Use T ₄In the similar experiment that-LNA carries out (accompanying drawing 34), depend on its higher stability of doing of inferring according to the higher stability of the LNA/DNA duplex of reporting, can expect that then opening of corresponding hair clip needs higher invert point.For verifying these hypothesis, replace 5 Nucleotide in dried with corresponding LNA monomer and synthesize and be similar to T ₄The T of-DNA ₄-LNA (is respectively T ^LWith ^MeC ^L).For the hair clip of modifying, only beginning from the hair clip to ssLNA changes can be observed and is higher than 80 ℃ (accompanying drawing 35A).T ₄-DNA and A ₄The mol mixture that waits of-DNA produces and independent T ₄The variation of the uniform temp that-DNA observes, but to A ₄-DNA/T ₄The variation of-DNA mixture, the increase of optical density(OD) (hyperpigmentation) is more strong.A is not depended in the hyperpigmented increase of mixture ₄The extra variation of-DNA hair clip, because separately oligonucleotide not have to surpass 20 ℃ variation, it is consistent with previous report, promptly VITAMIN B4 is compared with the thymus pyrimidine in encircling and is made the hair clip instability.Therefore pass through A ₄-DNA/T ₄The fusing of-DNA duplex has illustrated the hyperpigmentation that mixture increases best, has A although can not estimate its hairpin structure with it ₄T between the duplex structure of-DNA ₄The ratio that-DNA distributes.

Because T ₄The stability of-LNA hair clip and A ₄The unstable of-DNA hair clip estimates that these two oligonucleotide mixtures do not transform 20-80 ℃ of temperature range.Therefore locate to find that at 37.3 ℃ quite low hyperpigmented conversion is a difficult problem.By comparing A with the characteristic of carrying out the palindromic sequence of NMR broad research ₄-DNA/T ₄The specific conversion of-LNA mixture is understood best.For example, shown by duplex to have at 0.3mM at self complementary sequence 5 '-CGCGTTAACGCG that lower temperature forms, the variation of 33 ℃ hair clip and the variation of 48 ℃ random coil ( ^Vii).As accompanying drawing 36, schema 1 is described, and our system almost is identical, except hair clip forms oligonucleotide (T ₄-LNA) do not have self complementary sequence and need other oligonucleotide form duplex in ring zone.Required oligonucleotide (A ₄-therefore DNA) do not form hair clip and can not bring in room temperature and be used to explain A ₄-DNA/T ₄Any difficult problem of-LNA mixture melt.By the characteristic of palindromic sequence relatively, by the DNA/LNA duplex to A ₄-DNA and T ₄A has been explained in the conversion of the mixture of-LNA hair clip best ₄-DNA and T ₄-LNA mixture is 37.3 ℃ fusing.For palindrome oligonucleotide, people shown the conversion from the duplex to the hair clip be form that cruciform forms after producing by the initial ridge that is melted in the duplex center ( ^Viii).In case the formation cruciform then needs a little energy to prolong the fully separation of formed flexible connection up to two hair clips.We approve of and our situation similar mechanism and identical operations mechanism in the opposite direction, and the identical melting curve of fluctuating temperature model is obtained.These may hint the example that we have found that the chain that enters into highly stable LNA hair clip.

For 5 '-CGCGTTAACGCG sequence, by NMR lesser temps can observe hairpin structure and never observe by hair clip be converted into fully duplex ( ^Vii), it can show that the balance quenching temperature is lower than invert point.As if the quite low hyperpigmentation that transforms show conversion A ₄-DNA and T ₄The mixture of-LNA hair clip is incomplete for the DNA/LNA duplex.If these hint melting temperature(Tm)s are higher and approach the melting temperature(Tm) of LNA hair clip, then from the hair clip to the duplex, can transform more completely.In fact work as pyrene pseudonucleus thuja acid and be inserted in A ₄During the A4 zone of DNA middle, we have found this phenomenon (accompanying drawing 34) really.It has been found that in the past that the single INA in the duplex A/T zone inserted the remarkable increase that has caused melting temperature(Tm).Here also observe and have 44.7 ℃ melting temperature(Tm), and in addition, when with from A ₄When comparing, the DNA/LNA duplex of-DNA observes hyperpigmented remarkable increase (accompanying drawing 2A and 2D).Higher in this case hyperpigmentation shows A ₂PA ₂-DNA/T ₄-LNA duplex has the better ability that forms from the LNA hair clip transforms.

From above-mentioned discovery as can be known pyrene reduced the melting temperature(Tm) of the duplex that contains LNA in the insertion of LNA monomer relative position, we have measured T ₄Suitable insertion in the doing of-LNA hair clip can reduce its stability and make it incline to target A ₄-DNA.Because T ₄Can not measure to such an extent as to the invert point of-LNA is too high, people wish to observe the oligonucleotide P that single pyrene inserts ₂-LNA and P ₅-LNA is respectively in the thermal transition of 81.1 ℃ and 71.4 ℃, and the twice pyrene inserts oligonucleotide P-P LNA 69.1 ℃ thermal transition, and hyperpigmentation is lower than 3 conversion very much although it must be admitted that.This means should be very careful explanation and by utilizing bracket that this translation table is shown in the accompanying drawing 34.Do not consider whether to transform be since hair clip open or because the fusing of uncertain duplex, we insert the evidence that oligonucleotide may be easier to form with its complementary ssDNA target duplex with it as these pyrenes of supposition.

As oligonucleotide P with INA monomer P ²-LNA is inserted into after first dried Nucleotide, has A ₄The melting ratio of the duplex of-DNA (accompanying drawing 35B) has the A that inserts without any P ₄The T of-DNA ₄-LNA (accompanying drawing 35A) is observed remarkable intensive hyperpigmentation.Hyperpigmentation is to be shown in accompanying drawing 2A-C T for referencial use ₄-DNA/A ₄-DNA duplex hyperpigmented only about half of.Also as expected, because P has observed the increase of invert point to hybridizing the stabilization in DNA.For two invert points and hyperpigmentation, has the oligonucleotide P of the P insertion of the ring that is adjacent to its corresponding hair clip formation ⁵-LNA is found has similar result (accompanying drawing 35C).

Have two P and insert the P-P-LNA that produces doing the zone, its corresponding duplex with A4-DNA demonstrates even higher invert point, but more significantly, and hyperpigmentation is identical with the duplex of unmodified almost.These have proved obviously that P is inserted into can make the LNA of particular type be easier to target and increase stability with the duplex of target simultaneously among the LNA with secondary structure, the latter infers that carrying the LNA probe that P inserts has higher invert point.People attempt by inserting P to corresponding to the further stable duplex that has the LNA probe in the target in the zone of the ring of probe.From accompanying drawing 34 as seen, oligonucleotide A ₂PA ₂Shown in addition higher invert point and equally in the LNA probe that inserts two P (P-P-LNA) find the highest invert point.Stablizing duplex by the extra insertion in the target has also improved as A ₂PA ₂The hyperpigmentation that oligonucleotide embodied (accompanying drawing 35D).When formation has the duplex of P-P-LNA, (APA) ₂-DNA shows the highest melting temperature(Tm).In this case, melting temperature(Tm) is than the invert point (69.1 ℃) higher significantly (83.3 ℃) of P-P-LNA hair clip.

(a) Williams, J.C., Casa-Green, S.C., Mir, K.U. and Southern, E.M. (1994) is by hybridizing in the interaction of array research oligonucleotide: unsettled end is to the influence of duplex output.Nucleic?Acids?Res.，22，1365-1367.

(b) Milner, N., Mir, K.U. and Southern, E.M. (1997) selects effective antisense reactant by the combination oligonucleotide arrays.Nature?Biotechnol.，15，537-541.

(c) Southern, E., Mir, K.andShchepinov, the molecule on M. (1999) microarray is done mutually.Nature?Genet.，21，5-9.

^Ii.(a) Lockhart, D.J., Dong, H.B., Michael, C., Follettie, M.T.Gallo, M.V., Chee, M.S., Mittmann, M.Wang, C., Kobayashi, M., Horton, H. and Brown, E.L.

(1996) hybridize expression regulation in high density oligonucleotide array.Nature Biotech-nol., 14,1675-1680. (b) Cronin, M.T., Fucini, R.V., Kim, S.M., Masino, R.S., Wespsi, R.M. and Miyada, C.G. (1996) carries out cystic fibrosis sudden change detection by hybridizing in luminous dna probe array.Hum.Mutat.，7，244-255。

^Iii.Nguyen, H.-K. and Southern, E.M. (2000) simplifies the secondary structure of DNA target most by embedding modified deoxy nucleoside: the meaning of carrying out foranalysis of nucleic acids by hybridization.NucleicAcids?Res.，28，3904-3909。

^Iv(a) Christensen, U.B. and Pedersen, E.B. (2002) contain the insertion nucleic acid that 1-O-(1-pyrenyl methyl) glycerol is inserted: the discriminating of the stabilization of dsDNA and DNA and RNA.Nucl.Acids?Res.，30，4918-4925.

(b) Filichev, V.V. and Pedersen, E.B. (2003) has N-(1-pyrenyl methyl)-(3R, 4R)-4 insertion nucleic acid (INAs) of (methylol) pyrrolidyl-3-phenol insertion.DNA (RNA) duplex and DNA three-dimensional connection stability.Org.Biomol., during Chem. publishes.

^v.Petersen, M., Nielsen, C.B., Nielsen, K.E., Jensen, G.A., Bondensgaard, K., Singh, S.K., Rajwanshi, V.K., Koshkin, A.A., Dahl, B.M., Wengel, J. and Jacobsen, the conformation (LNA) of J.P. (2000) sealing nucleic acid.J.Mol.Rec-ogn.，13，44-53。

^Vi.Vallone, P.M., Paner, T.M., Hilario, J., Lane M.J., Faldasz, B.D. and Be-night, the fusing of A.S. (1999) short dna hair clip research: ring sequence and in abutting connection with the Biopolymer. that influences of base pair identity to the hair clip thermodynamic stability, 50,425-442.

^Vii.Hald, M., Pedersen, J.B., Stein, P.C., Kirpekar, F.Jacobsen, the comparison of the hair clip stability of J.P. (1995) palindrome d (CGCG (A/T) 4CGCG) oligonucleotide.Nucleic?Acids?Res.，23，4576-4582。

^Viii.Wemmer, D.E., Chou, S.H., Hare, D.R. and Reid, the duplex among B.R. (1985) DNA-hair clip transforms: to the NMR research of CGCGTATACGCG.NucleicAcids?Res.，13，3755-3772。

Embodiment 26

The preparation of intercalator pseudonucleus thuja acid

Present embodiment described N-(1-pyrenyl methyl)-(3R, 4R)-preparation of 4-(methylol) pyrrolidyl-3-phenol (4) with and application in INAs is synthetic and to INA/DNA, the research of the hybridization avidity in INA/RNA duplex and DNATWJ zone.

When inserting N-(1-pyrenyl methyl) azasugar when protruding, then observe the stability of the increase that good discriminating between the stability of INA/DNA and INA/RNA duplex and DNA three-dimensional connect.

From the synthetic 1 '-azacytidine pyrenyl methyl pseudonucleus thuja acid 4 of the pure 1-azacytidine analogue of the optical siomerism of the own furanose 1 of 2-deoxidation-D-or 2-deoxidation-D-ribofuranose 2. ¹³The pyrene substrate with chloromethyl and formaldehyde function of

secondary amine

1 and 2 of can being coupled is used (accompanying drawing 37).

The phosphoramidate 6 of DMT protection is that oligonucleotide is synthetic required.Handle primary alconol 4 and carry out further purifying with excessive DMTCI in pyridine and produce compound 5 with 61% productive rate by silicagel column.By in excessive Hunig base ¹²With 2-cyanoethyl-N, N-sec.-propyl chloro phosphoramidate handles to synthesize the not success of final phosphoramidate under the situation about existing.For obtaining required phosphoramidite 6, we use the selectable 2-of utilization cyanoethyl-N, N, N ', N '-tetraisopropylphosphane and N, the method for N-diisopropylammoniumtetrazolide ¹⁵Productive rate with 57% obtains compound 6.

Twice of coupling time (24 minutes) that increases by automatic solid phase dna synthesizer utilization and recirculation are incorporated into phosphoramidite 6 in the different oligonucleotide sequences and produce INAs, compare with about 99% (coupling in 2 minutes) of commercial phosphoramidite, the coupling efficiency of pyrene azasugar derivative 6 is about 80-85%

The hybridization (accompanying drawing 38) that synthetic INAs is used to INA/DNA and INA/RNA duplex is connected the research of (TWJ) (accompanying drawing 39) with the INA/DNA three-dimensional.

Each that has that integration N-(1-pyrenyl methyl) azasugar causes ssDNA as the INA of ridge is modified and is reduced by 1.2 ℃ melting temperature(Tm) (accompanying drawing 15).The corresponding of deoxynucleotide (dG) of containing a ridge has significantly reduced Tm=32.2 ℃ (ATm=-10.8 ℃) with reference to duplex.For the INA/RNA duplex, compare with the duplex (A item) of coupling fully, contain sequence C and reduced the stability of 10 ℃ of INA/RNA duplexs and 9.6 ℃ with reference to the pyrene of B respectively.Therefore, have pyrene azasugar integrate INA as ridge have than with ssRNA preferably with the avidity of complementary ssDNA hybridization.The difference of ssDNA and ssRNA melting temperature(Tm) is seemingly owing to pyrene partial amt institute among the target ODN is additional.These results are also consistent with insertion 1-O-(1-pyrenyl methyl) twice research as ridge of glycerol.The bigger difference of observing INA/DNA and INA/RNA then is up to 25.8 ℃.8 in such cases, and to compare the INA/DNA structure stabilized with the wild-type duplex, and wherein the wild-type duplex is opposite with our situation, and its Tm has slight reduction.The flexibility of ridge may be an important factor that obtains two duplex stabilizations and difference.Synthesizing of different joints and planar aromatic series part is also underway.The shown difference of RNA/DNA can be used to purifying or detection and have DNA target in the mixture of very identical RNA sequence.

When pyrene azasugar intercalator is inserted among the INA (F3), be connected (TWJ) (accompanying drawing 39) by two with DNA three-dimensional that the arm that involvement connects is formed, with the ODN (F2) that has dA in same position or the ODN (F1) that does not have to insert compare be found cause suitable stable.Undoubtedly the hybridization in the arm is important for the stability of complex body; Our preparation has the ODNs (E2 and E3 item) of the mispairing in the arbitrary arm of TWJ.It causes hybridizing the reduction greatly of avidity in both cases

Experiment

Conventional

During by Bruker AC-300 FT NMR spectrometer record 300MHz ¹When H NMR and 75.5MHz ¹³The NMR spectrum of C NMR. ¹The used international standard of H NMR spectrum is for being used for CDCl ₃, CD ₃The TMS of OD (δ .0.00); ¹³CDCl among the C NMR ₃(δ .77.0), CD ₃OD (δ .49.0).Kraton MS-50-RF by equipment FAB source carries out accurate mass of ion mensuration.Utilization is available from the quasi-molecular ions coupling [M+Hr] of glycerine matrix ⁺Ion.Utilization is available from TLC plates 60 F of Merck ₂₅₄Carry out thin-layer chromatography (TLC) analysis and in UV-lamp, use the triketohydrindene hydrate developer (at 100cm ³Butanols and 3cm ³0.3g triketohydrindene hydrate among the HOAc) video picture (254 and/or 343nm) azasugars and derivative thereof

The silica gel (0.063-0.200) that is used for column chromatography is available from Merck.By the synthetic ODNs of Assembler GeneSpecial DNA-Synthesizer (Pharmacia Biotech).Utilize WatersDelta Prep 4000 Preparative Chromatography systems to come purifying 5 '-O-DMT-ketone and 5 '-O-DMT-off ODNs.By the VoyagerElite Biospectrometry Research Station that the utilizes PerSeptive Biosystems ODNs that modifies that carried out the MALDI-TOF analytical proof.The all solvents of distillation before using.Used reagent is available from Aldrich, Sigma or Fluka.The reagent that is used for Gene Assembler is available from Cruachem (UK).

N-(1-pyrenyl methyl)-(3R, 4R)-4-[(1S)-1, the 2-dihydroxy ethyl] pyrrolidyl-3-alcohol (3)

Method A

(50mg 0.34mmol) is dissolved in DMF (5cm with Azasugar 1 ³), 1-(chloromethyl) pyrene (103mg, 0.41mmol) and add Et3N (0.057cm ³, 0.41mmol) reaction mixture stirs under the room temperature in nitrogen and spends the night.Solvent evaporated under reduced pressure and with toluene (2 * 5cm ³) coevaporation together.By using CH ₂Cl ₂(0-20% v/v) carries out chromatography as the silicagel column of elutriant to resistates and obtains pure product 3 (70mg, 57%): R/MeOH _f0.20 (10%MeOH/CH ₂Cl ₂); δ _H, (CD ₃OD) 2.36 (1H, m, H-4), 2.95 (1H, m, H-5), 3.08 (1H, dd, J 2.8 and 10.5, H-2), 3.22 (1H, dd, J 5.4 and 12.0, H-5), 3.34 (1H, m, H-2), 3.50-3.65 (3H, m, CH[OH] CH ₂OH), 4.42 (1H, m, H-3), 4.65 (2H, s, CH ₂Pyrenyl-1-yl), 4.88 (3H, br.s, 3xOH), 7.90-8.40 (9H, m, H _Arom); δ _C(CD ₃OD) 50.7 (C-4), 56.7 (C-5), 57.2 (C-2), 62.7 (CH2 pyrenyls-1-yl), 65.7 (CH ₂OH), 71.8 (CH[OH]), 72.7 (C-3), 123.8,125.5,125.8,125.9,126.6,126.8,127.3,128.0,128.2,129.1,129.4,129.9,131.2,131.9,132.5,133.2 (pyrenyls-1-yl); M/z (FAB) 362.1748[M+H] ⁺, C ₂₃H ₂₄NO ₃Need 362.1756.

Method B with Azasugar 1 (70mg, 0.48mmol) be dissolved in DMF/EtOH (3: 1,10cm ³) and 1-pyrenyl formaldehyde (270mg, 1.18mmol) and add NaCNBH ₃(74mg, 1.18mmol).Under the room temperature in nitrogen stirred reaction mixture spend the night.Add spissated HCl up to pH＜2.Solvent evaporated under reduced pressure is with toluene (2 * 5cm ³) coevaporation together.By using CH ₂Cl ₂(0-20% v/v) carries out purifying as the silicagel column of elutriant to resistates and obtains compound 3 (110mg, 63%)/MeOH.

N-(1-pyrenyl methyl)-(3R, 4R)-4 (methylol) pyrrolidyl-3-alcohol (4)

Method A.

(100mg 0.86mmol) is dissolved in DMF (10cm with Azasugar 2 ³) and 1-(chloromethyl) pyrene (257mg, 1.03mmol) and add Et3N (0.140cm ³, 1.03mmol).Under the room temperature in nitrogen stirred reaction mixture spend the night.Solvent evaporated under reduced pressure and with toluene (2 * 5cm ³) coevaporation together.Resistates is dissolved in H ₂O/CH ₂Cl ₂(1: 1,40cm ³) and use CH ₂Cl ₂Extract water layer.Dry (Na ₂SO ₄) the blended organic constituent, vacuum-evaporation and by using CH ₂Cl ₂(0-20%, v/v) carry out chromatography as the silicagel column of elutriant provides the compound 4 in the title (130mg, 46%): R to/MeOH _f, 0.17 (10%MeOH/CH ₂Cl ₂); δ _H(CDCl ₃) 2.08 (1H, m, H-4), 2.29 (1H, m, H-5), 2.57 (1H, dd, J 2.8 and 10.2, H-2), 2.85 (2H, m, H-2 and H-5), 3.43 (2H, s, CH ₂OH), 3.47 (2H, br.s, 2xOH), 4.08 (1H, m, H-3), 4.19 (2H, s, CH ₂Pyrenyl-1-yl), 7.90-8.40 (9H, m, H _Arom); δ _C(CDCl ₃) 49.8 (C-4), 55.9 (C-5), 57.5 (C-2), 62.3 (CH2 pyrenyls-1-yl), 64.2 (CH ₂OH), 73.9 (C-3), 123.3,124.4,124.6,124.8,125.1,125.9,127.3,127.6,127.8,129.5,130.7,130.9,131.1 (pyrenyls-1-yl); M/z (FAB) 332.1631[M+H] ⁺, C ₂₃H ₂₄NO ₃Require 332.1651.

Method B. with Azasugar 2 (1.18g, 10.1mmol) be dissolved in DMF/EtOH (3: 1,150cm ³) and 1-pyrene-formaldehyde (3.47g, 15.1mmol) and add NaCNBH ₃(950mg, 15.1mmol).Reaction mixture stirs under the room temperature in nitrogen and spends the night.Add spissated HCl up to pH＜2.Solvent evaporated under reduced pressure and with toluene (2 * 5cm ³) coevaporation together.Resistates is dissolved in H ₂O/CH ₂Cl ₂(1: 1, v/v, 150cm ³) and use CH ₂Cl ₂(3 * 75cm ³) the extraction water layer.Organic constituent (the Na of dry mixed ₂SO ₄), reduction vaporization.Utilize CH ₂Cl ₂(0-20%, v/v) carry out silica gel column chromatography chromatography purification resistates provides that crystallization is buttery compound 4 (1.9g, 57%) when leaving standstill to/MeOH, mp 104-105 ℃.

Method C. will be in EtOH compound 3 (110mg, cooling solution 0.304mmol) under agitation is added to NalO ₄(71.6mg is in aqueous solution 0.335mmol).Add NaBH after 30 minutes ₄(12.3mg, 0.335mmol).After 30 minutes, it is 2 up to pH that the solution of generation carries out violent stirring with the 2MHCl acidifying.Remove in the vacuum and desolvate.Resistates is dissolved in H ₂O/CH ₂Cl ₂(1: 1, v/v, 20cm ³) and use CH ₂Cl ₂(4 * 15cm ³) extract.Organic layer (the Na of dry mixed ₂SO ₄), evaporation drying provides compound 4 (40mg, 40%) under the low pressure.

N-(1-pyrenyl methyl)-(3R, 5R)-4-[(4,4 '-dimethyl triphenyl methoxyl group) methyl] pyrrolidyl-3-alcohol (5)

(139mg 0.42mmol) is dissolved in anhydrous pyridine (10cm with compound 4 ³) and add DMTCl (178mg, 0.53mmol).Room temperature stirs the mixture 24 in nitrogen.Add MeOH (1cm ³) inhibited reaction and solvent evaporated under reduced pressure and usefulness toluene (2 * 5cm ³) coevaporation.Resistates heavily is dissolved in H ₂O/CH ₂Cl ₂(1: 1, v/v, 20cm ³), and use saturated NaHCO ₃The solution washing mixture.Organic layer (the Na of dry mixed ₂SO ₄), and concentrating under reduced pressure.Utilize the silica gel column chromatography chromatography (the 5-40%EtOAc/ hexanaphthene, v/v) purifying produces the compound 5 in the title, it is that a kind of foam (160mg, 61%) is used for step thereafter and need not be further purified: R _f, 0.45 (49%EtOAc/49% hexanaphthene/2%Et3N, v/v/v); δ _H(CDCl ₃) 2.20 (1H, m, H-4), 2.34 (1H, m, H-5), 2.53 (1H, br.s, OH), 2.62 (1H, dd, J5.6 and 9.9, H-2), 2.72 (1H, dd, J2.5 and 9.8, H-2), 3.06 (3H, m, CH ₂ODMT and H-5), 3.71 (6H, s, OCH ₃), 4.01 (1H, m, H-3), 4.21 (2H, s, CH ₂Pyrenyl-1-yl), 6.78 (4H, m, DMT), 7.10-7.40 (9H, m, DMT), 7.90-8.40 (9H, m, H _Arom); δ _C(CDCl ₃) 48.8 (C-4), 55.2 (OCH ₃), 56.1 (C-5), 58.0 (C-2), 61.9 (CH2 pyrenyls-1-yl), 64.5 (CH ₂OH), 74.9 (C-3), 85.9 (C-Ar ₃), 113.0,123.8-132.3 (DMT and pyrenyl-1-yl), 144.9,158.4 (DMT); M/Z (FAB) 634.2740[M+H] ⁺, C ₄₄H ₄₂NO ₅Need 634.2722.

N-(pyrenyl-1-ylmethyl)-(3R, 4R)-3-O-[2-cyanoethyl oxygen (diisopropylaminoethyl)-Ya phosphorus base]-4-[4,4 '-dimethyl triphenyl methoxyl group] methyl] tetramethyleneimine (6)

(140mg 0.22mmol) is dissolved in anhydrous CH with compound 5 in nitrogen ₂Cl ₂(5cm ³) in.Add N, (61mg 0.42mmol) drips 2-cyanoethyl-N to N-diisopropylaminoethyl tetrazolium then, N, N, N, N ', N '-tetra isopropyl phosphorus (0.140cm ³, 0.44mmol).2.0h analyzing, back TLC shows do not had parent material and used H ₂O (1cm ³) add CH then ₂Cl ₂(10cm ³) inhibited reaction.Use saturated NAHCO ₃The aqueous solution (2 * 10cm ³) purging compound.Dry organic phase (Na ₂SO ₄) and removal of solvent under reduced pressure.(0-20% v/v) carries out silica gel column chromatography chromatography purification resistates to utilize hexanaphthene/EtOAc.The active component of vacuum-evaporation blended UV-provides foamed composition 6 (158mg, 57%), its be used for ODN synthetic before with anhydrous acetonitrile (3 * 30cm ³) coevaporation together.R _f0.85 (49%EtOAc/49% hexanaphthene/2%Et ₃N, v/v/v); δ _H(CDCl ₃) 0.93 (6H, m, CH ₃[Pr _i]), 1.04 (6H, m, CH ₃[Pr ⁱ]), 2.30 (2H, m, H-4 and H-5), 2.48 (2H, m, CH ₂CN), 2.64 (1H, m, H-2), 2.78 (1H, m, H-2), 2.98 (2H, m, OCH ₂CH ₂CN), 3.08 (1H, m, H-5), 3.50 (4H, m, CH[Pr ⁱ] and CH ₂ODMT), 3.65 (6H, s, OCH ₃), 4.01 (1H, m, H-3), 4.20 (2H, m, CH ₂Pyrenyl-1-yl), 6.68 (4H, m, DMT), 7.05-7.40 (9H, m, DMT), 7.85-8.40 (9H, m, H _Atom); (CDCl ₃) 148.2 (s), 149.0 (s) ratio 2: 1.

Synthetic and the purifying of that modify and oligodeoxyribonucleotide unmodified

Utilize commercially available 2-cyanoethylphosphoramidites and compound 6 by Pharmacia Gene Assembler Special synthesizer with the synthetic oligodeoxyribonucleotide of the scale of 0.2 μ mol (every circulation pack into 7.5 μ mol, Pharmacia primer).The synthetic regular flow process of following dna synthesizer.Be increased to 24 minutes and circulation is repeated twice with 6 coupling time from 2.From solid support separate 5 '-O-DMT-ketone ODNs and at 55 ℃ with 32% NH ₃Solution (1cm ³) go to protect 24h to utilize Hamilton PRP-1 post to carry out purifying by preliminary HPLC then.Solvent system is buffer A [950cm ³0.1M NH ₄HCO ₃And 50cm ³CH ₃CN (pH=9.0)] and buffer B [250cm ³0.1 MNH ₄HCO ₃And 750cm ³CH ₃CN (pH=9.0)), it uses in the following order: the linear gradient of 0-70%B among 5 minutes A, 30 minutes A, the linear gradient of 5 minutes 70-100%B in A.Flow velocity is 1cm ³

Min

^-15 '-O-DMT-ketone ODNs wash-out of purifying is a peak [UV contrast 254nm and 343nm (being used to contain the pyrene of ODNs)] after about 30 minutes.

Component is used 10% HOAc (1cm then by vacuum concentration ³) solution-treated 20 minutes and under identical condition, be further purified the trityl removal ODNs that is provided at 23-28 minute wash-out by HPLC.By preliminary HPLC, the purity of synthetic oligonucleotide is 99-100%.Solution that vacuum concentration produced and water coevaporation are removed volatile salt for twice ODNs are provided, and it is used to the mensuration of melting temperature(Tm).All oligonucleotide that contain the pyrenylmethylazasugar derivative are analyzed by MALDI TOF confirms (C item: found 4005.65, calcd.4005.76; The D item: 4398.02, calcd.4398.87; F3 item: found 4903.05, calcd.4904.89).

The experiment of unwinding

Measure melting temperature(Tm) by the Perkin-ElmerUV/VIS spectrometer that PTP-6 Peltier temperature-program element is housed.Measure the light absorption ratio from 18 ℃ to 85 ℃ of 1cm cell at 260nm.Measure melting temperature(Tm) as the maximum value of drawing the derivative melting curve.Oligodeoxyribonucleotide is dissolved in moderate salt buffer (pH=7.0,1mM EDTA, 10mMNa2HP04 * 2H20,140mMNaCl) concentration of the every chain 1.0 μ M of formation.

Embodiment 27

Fluorescence when hybridizing in the mispairing target

Fluorescent quenching is the strong interactional signal of fluorophore and duplex.Construction minimizes is calculated and has been supported that wherein pyrene partly is inserted in the duplex.Therefore can expect that contiguous importing of inserting the mispairing in site has caused the adaptive increase of pyrene and therefore increased fluorescence.These also be found do not consider intercalator and the mispairing (table 6) that imports.

Table 6: hybridize in complementary sequence or hybridize in the fluorescence data of the mono-modified ODN of one of 6 adjacent simple point mutation types of difference.

5′-A-G-C-T-?T-Z-Y-T-T-G-A-G-3′

Title	?Z	?Y	382nm Rel. intensity	395nm Rel. intensity	480nm. Rel. intensity
Title	?Z	?Y	382nm Rel. intensity	395nm Rel. intensity	480nm. Rel. intensity	Independent probe	?--	?--	?48	?40	?1
?Wt	?G	?C	?15	?12	?1	Independent probe	?--	?--	?48	?40	?1
?Wt	?G	?C	?15	?12	?1	?Mut.1	?C	?C	?59	?50	?2
?Mut.2	?A	?C	?75	?63	?2	?Mut.1	?C	?C	?59	?50	?2
?Mut.2	?A	?C	?75	?63	?2	?Mut.3	?T	?C	?50	?42	?2
?Mut.4	?G	?T	?34	?29	?2	?Mut.3	?T	?C	?50	?42	?2
?Mut.4	?G	?T	?34	?29	?2	?Mut.6	?G	?A	?63	?53	?2

Probe III can be used for the hypothesis that simple point mutation detects for checking, and it is hybridized in the target with all 4 variants of Y, and (intensity at the excimer band at 480nm place in the time of T) increases (table 7) significantly for Y=G, A when introducing mispairing.People expect once more by introducing mispairing, also can 382 and the 395nm place observe the fluorescence band.Surprisingly, also increased along with importing the fluorescence of mispairing at 480nm place in 3 ' one sides of two intercalators, this shows that two pyrenes partly can (table 7) interact with each other.These are only expected to be wide enough so that the marquis produced when the pyrene part was interactional if encircle.It should be noted that when hybridizing in complementary sequence, 382 and the fluorescent quenching at 395nm place, but fluorescence increases when having the sequence of a mispairing for one when hybridizing.Therefore using all three wavelength (382,295 and 480) to distinguish fully-complementary sequence should be possible with the complementary sequence with a mispairing.

Table 7: hybridize in the complementary sequence sequence or hybridize the fluorescence data of modifying ODN in the twice of one of 6 different simple point mutations.

5′-A-G-C-T-T-Z-Y-T-T-G-A-G-3′

Title	?Z	?Y	382nm Rel. intensity	395nm Rel. intensity	480nm Rel. intensity
Title	?Z	?Y	382nm Rel. intensity	395nm Rel. intensity	480nm Rel. intensity	?SsDNA	?--	?--	?44	?38	?17
?Wt	?G	?C	?19	?17	?4	?SsDNA	?--	?--	?44	?38	?17
?Wt	?G	?C	?19	?17	?4	?Mut.1	?C	?C	?84	?73	?14
?Mut.2	?A	?C	?74	?64	?10	?Mut.1	?C	?C	?84	?73	?14
?Mut.2	?A	?C	?74	?64	?10	?Mut.3	?T	?C	?84	?74	?12.5
?Mut.4	?G	?T	?62	?54	?8	?Mut.3	?T	?C	?84	?74	?12.5
?Mut.4	?G	?T	?62	?54	?8	?Mut.5	?G	?G	?84	?74	?17
?Mut.6	?G	?A	?70	?60	?12	?Mut.5	?G	?G	?84	?74	?17

Embodiment 28

Thermodenaturation research

When comparing, insert intercalator and in DNA, be accompanied by the specific decline of hybridizing with fully-complementary sequence with the sequence that around intercalator, has base-pair mismatch.Whether the purpose of experiment is to detect has pyrene and partly inserts, the mispairing of the both sides of intercalator, be adjacent to and intercalator between duplex also belong to this situation.Measure specificity by the difference of measuring complete complementary duplex and wherein importing the melting temperature(Tm) between the duplex of a mispairing.The melting temperature(Tm) data presentation is in table 8.

Table 8: have the different melting temperature(Tm) data of inserting the ODNs of patterns of hybridizing in three possible point mutation of complementary strand or Nucleotide #6. and #7.X=1

5 ' A-G-C-T-T-Z-Y-T-T-G-A-G target

3′T-C-G-A-A-C-G-A-A-C-T-C????????????????????????????????????????????????I

The target probe

Z???Y???I??????????????II?????????????III????????????IV????????????V

[℃]???????????[℃]???????????[℃]???????????[℃]??????????[℃]

Wt?????G???C???47.4???ΔT?????50.4???ΔT?????51.4???ΔT?????45.4???ΔT????60.8???ΔT

Mut.1??C???C???23.4???-24.0???34.0???-16.0???38.0???-13.4???23.4???-22.0??33.8???-27.0

Mut.2??A???C???30.8???-16.6???34.2???-16.2???36.6???-14.8

Mut.3??T???C???27.6???-18.8???33.6???-16.8???35.2???-16.2???25.4???-20.0??37.4???-23.4

Mut.4??G???T???36.2???-11.2???42.2???-8.2????45.2???-6.2????36.6???-8.8???45.8???-15.0

Mut.5??G???G???40.0???-7.4????42.4???-8.0????38.6???-12.8???39.4???-6.0???53.2???-7.6

Mut.6??G???A???39.8???-7.6????39.0???-11.4???39.0???-12.4???39.2???-6.2???49.0???-11.8

As seen from Table 8, opposing is modified the specificity of mispairing of probe within the scope of unmodified probe specificity, although the selectivity of terminal C-C mispairing looks the (table 8 that descended with inserting site 5 '; The Mut.1 of probe I I and III).The tendency of other unique unanimity is that wherein two intercalators are resisted the mispairing of two Nucleotide away from any insertion site by the probe of 4 separated double modifications of Nucleotide (probe V) more specifically than the probe (probe 1) of unmodified.Remaining melting temperature(Tm) difference approaches the value of unmodified duplex.Research the most important thing is that probe I II is optionally to the target with the suitable base between the pyrene part about the simple point mutation type, and three may mispairing outer two have more specificity, and last a kind of situation has less specificity (table 8).

Embodiment 29

Beacon-primer

The example of beacon-design primer is presented in the accompanying drawing 40.Primer comprises 39 Nucleotide, and it is designed such that they can produce stem-ring structure.

Primer has a target-complementary region, also promptly is complementary to target DNA, and its 24 Nucleotide are long.In addition, primer has self complementary zone, also promptly can hybridize another end in primer.

Self complementary zone is that 15 Nucleotide are long and contain other 4 intercalator pseudonucleus thuja acids.Two intercalator pseudonucleus thuja acids are placed and make it can form a kind of intramolecularly excimer.

The melting temperature(Tm) of primer/target crossbred is about 67 ℃, and the melting temperature(Tm) that self hybridizes is about 46 ℃.

The beacon primer can be used for PCR and can be used for quantitative PCR.

Target-specific beacon primer and template DNA are provided (accompanying drawing 41A).Beacon primer and template DNA are stored in 0-4 ℃.

Double-stranded DNA is at 94 ℃ of sex change (accompanying drawing 41B).The beacon primer annealed (accompanying drawing 41C) with target DNA and by Taq polymerase extension primer at about 65 ℃.

Subsequently, temperature reduce to about 45 ℃ and not hybridization the beacon primer by self hybridization (accompanying drawing 41D).Measure excimer fluorescence and be scaled the amount of the beacon primer of extension.

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Claims

1. the intercalator pseudonucleus thuja acid that has following formula

X-Y-Q

Wherein X be for can insert the nucleic acid of following general formula or the backbone monomers unit of nucleic acid analog main chain,

N=1 to 6 wherein

R ₁Be the phosphorus atom of trivalent or pentavalent replacement,

R ₂Be independently selected from the atom that can form at least two chemical bonds, R ₂Randomly replaced separately, and R ₆It is a protecting group;

Q is the intercalator of the conjugate system that comprises that at least one is generally planar, and it can be piled up altogether with the nuclear base of DNA; And

Y is with the unitary arbitrary R of described backbone monomers ₂The shank that is connected with described intercalator; And

Wherein the total length of Q and Y at 7  in the scope of 20 ,

Condition is when intercalator is pyrene, the total length of Q and Y at 9  in the scope of 13 .

2. the nucleotide analog of claim 1, wherein the backbone monomers unit can so that at the most 6 atoms, mode that 2 phosphorus atom of main chain are separated insert the phosphate backbone of nucleic acid or nucleic acid analog, two the most close intercalators of phosphorus atom of described main chain.

3. the intercalator pseudonucleus thuja acid of claim 1, wherein the backbone monomers unit is selected from and comprises that at least one is selected from the backbone monomers unit of following chemical group: phosphoric acid salt, phosphoric acid ester, phosphodiester, phosphoramidate, phosphoro chloroamidite, phosphorp diamidite and phosphoramidit.

4. according to the intercalator pseudonucleus thuja acid of claim 3,6 atoms are long at the most wherein can to form the chemical bonding of atom of key from least one phosphorus atom with contiguous Nucleotide at least one.

5. the intercalator pseudonucleus thuja acid of claim 1, wherein the backbone monomers unit is selected from acyclic backbone monomers unit.

6. the intercalator pseudonucleus thuja acid of claim 1, wherein the backbone monomers unit is selected from and can stablizes the acyclic backbone monomers unit that protrudes inset.

7. claim 3 and 4 each intercalator pseudonucleus thuja acids, wherein the backbone monomers unit comprises a phosphoramidit.

8. claim 3 and 4 each intercalator pseudonucleus thuja acids, wherein the backbone monomers unit comprises the pentavalent phosphoramidate.

9. claim 3 and 4 each intercalator pseudonucleus thuja acids, wherein the backbone monomers unit comprises trivalent phosphoramidit.

10. the intercalator pseudonucleus thuja acid of claim 1; wherein the backbone monomers unit comprises the protecting group that can remove, and wherein the removal of protecting group can make the chemical reaction between intercalator pseudonucleus thuja acid and Nucleotide and/or nucleotide analog and/or another intercalator pseudonucleus thuja acid become possibility.

11. the intercalator pseudonucleus thuja acid of claim 10, wherein protecting group can be removed by acid treatment.

12. the intercalator pseudonucleus thuja acid of claim 10; wherein protecting group is selected from trityl, mono methoxy trityl; 2-chlorine trityl; 1; 1,1,2-tetrachloro-2; two (right-the p-methoxy-phenyl)-ethan (DATE) of 2-, 9-phenyl yanthine-9-base (pixyl) and 9-(right-p-methoxy-phenyl) xanthine-9-base (MOX).

13. the intercalator pseudonucleus thuja acid of claim 10, wherein protecting group be selected from 4,4 '-dimethoxytrityl methoxyl group and dimethoxytrityl (DMT).

14. the intercalator pseudonucleus thuja acid of claim 1, wherein intercalator comprises the chemical group that is selected from polyaromates and heteropolyaromates.

15. the intercalator pseudonucleus thuja acid of claim 1, wherein intercalator is selected from polyaromates and heteropolyaromates.

16. the intercalator pseudonucleus thuja acid of claim 1, wherein intercalator is selected from phenanthroline, azophenlyene, phenanthridines, anthraquinone, pyrene, anthracene, cyclic hydrocarbon, phenanthrene, picene, chrysene, naphtacene, dihydroketoacridine, benzanthrene, 1-2-toluylene, oxalyl pyrido-carbazole, phenylazide, porphyrin and psoralene.

17. the intercalator pseudonucleus thuja acid of claim 1, wherein intercalator is a pyrene.

Another end of the chain is connected to the backbone monomers unit 18. the intercalator pseudonucleus thuja acid of claim 1, its center tap comprise a m atomchain that is selected from C, O, S, N, P, Se, Si, Ge, Sn and Pb, and its medium-chain one end is connected to intercalator.

19. the intercalator pseudonucleus thuja acid of claim 18, wherein m is from 1 to 7 integer.

20. the intercalator pseudonucleus thuja acid of claim 18, its medium chain is replaced by the atom that one or more is selected from C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.

21. the intercalator pseudonucleus thuja acid of claim 1, its center tap is the azepine alkyl, oxa alkyl, thia alkyl or alkyl chain.

22. the intercalator pseudonucleus thuja acid of claim 1, its center tap is for being selected from the alkyl chain that C, H, O, S, N, P, Se, Si, Ge, Sn and Pb replace by one or more.

23. the intercalator pseudonucleus thuja acid of claim 1, its center tap is the ring texture that comprises the atom that is selected from C, O, S, N, P, Se, Si, Ge, Sn and Pb.

24. the intercalator pseudonucleus thuja acid of claim 23, its center tap is replaced by the atom that one or more is selected from C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.

25. the intercalator pseudonucleus thuja acid of claim 111, wherein intercalator pseudonucleus thuja acid is selected from (S)-1-(4,4 '-dimethoxytrityl methoxyl group)-3-pyrene methoxyl group-2-propyl alcohol and (R)-1-(4,4 '-the dimethoxytrityl methoxyl group)-3-pyrene methoxyl group-2-propyl alcohol.

26. each the method for intercalator pseudonucleus thuja acid of a synthetic claim 11 to 25, described method comprises the steps:

A1) provide and comprise the compound that intercalator and optional is connected to the shank of active group, described intercalator comprises at least a generally planar conjugate system, and it can be piled up altogether with the nuclear base of nucleic acid; With

C1) thus the reaction of described intercalator and described tab precursor obtains intercalator-joint; With

D1) provide one to comprise at least two active groups and randomly comprise the backbone monomers precursor unit of a shank, described two active groups can randomly be protected respectively and/or shelter); With

E1) described intercalator-joint and described backbone monomers precursors reaction and acquisition intercalator-joint-backbone monomers precursor; Perhaps

A2) provide one to comprise at least two active groups and randomly comprise the backbone monomers precursor unit of a shank, described two active groups can randomly be protected respectively and/or shelter); And

C2) thus described monomer precursor unit is obtained main chain-joint with the reaction of described tab precursor; And

D2) provide one to comprise intercalator and randomly be connected to the compound of the shank of active group, described intercalator comprises at least one generally planar conjugate system, and it can be piled up altogether with the nuclear base of nucleic acid; With

E2) described intercalator and described main chain-joint reaction and acquisition intercalator-joint-backbone monomers precursor;

Perhaps

A3) provide one to comprise that intercalator and is connected to the compound of the shank of active group, described intercalator comprises the conjugate system that at least one is generally planar, its can with the nuclear base and the common accumulation of nucleic acid; With

B3) provide the backbone monomers precursor unit that comprises at least two active groups and a shank, described two active groups can randomly be protected respectively and/or shelter; And

C3) reaction of described intercalator-shank and described backbone monomers precursor joint and acquisition intercalator-joint-backbone monomers precursor;

And

G) provide a phosphorescent substance that comprises the compound that two pseudonucleus thuja acids, Nucleotide and/or nucleotide analogs can be coupled together; And

I) obtain an intercalator pseudonucleus thuja acid.

27. the method for claim 0, wherein the intercalator active group is an electrophilic reagent.

28. the method for claim 0, wherein the intercalator active group is selected from alkylhalide group, mesyloxy alkyl and tosyl group oxygen base alkyl.

29. the method for claim 0, wherein cyclic or acyclic alkane are polysubstituted alkane.

30. the method for claim 0, its center tap active group is a nucleophilic reagent.

31. the method for claim 0, its center tap active group is a hydroxyl.

32. the method for claim 0, one or more joint active group is protected by a protecting group.

33. the method for claim 0, wherein at least one joint active group is 2,2-dimethyl-1,3-dioxalane-4-methyl alcohol.

34. the method for claim 0, wherein at least one joint active group is (S)-(+)-2,2-dimethyl-1,3-dioxalane-4-methyl alcohol.

35. the method for claim 0, wherein at least one joint active group is (R)-(+)-2,2-dimethyl-1,3-dioxalane-4-methyl alcohol.

36. the method for claim 262626 is a phosphitylating reagent comprising the reagent of phosphorescent substance.

37. the method for claim 26 is phosphordiamidite or chloro amido phosphoric acid ester comprising the reagent of phosphorescent substance.

38. the method for claim 36, wherein phosphitylating reagent is NC (CH ₂) ₂OP (Npr ⁱ ₂) ₂

39. the method for claim 0, wherein intercalator is a 1-pyrene Methochloride.

40. one kind is synthesized and comprises the oligonucleotide of at least a intercalator pseudonucleus thuja acid or the method for oligonucleotide analogs, wherein said comprising the steps:

I) each intercalator pseudonucleus thuja acid of claim 1 to 25 is contacted with the chain of the upholder bonded Nucleotide, oligonucleotide, nucleotide analog or the oligonucleotide analogs that are increasing; And

Ii) with described intercalator pseudonucleus thuja acid and described upholder bonded Nucleotide, oligonucleotide, nucleotide analog or oligonucleotide analogs reaction; And

Iii) randomly by increasing one or more Nucleotide, nucleotide analog or intercalator pseudonucleus thuja acid to further described oligonucleotide or the oligonucleotide analogs of prolonging of the oligonucleotide analogs of expectation sequence; And

Iv) described oligonucleotide or oligonucleotide analogs are separated with described solid support; With

Thereby v) obtain to comprise the oligonucleotide or the oligonucleotide analogs of at least one intercalator pseudonucleus thuja acid.

41. one comprises the oligonucleotide or the oligonucleotide analogs of the intercalator pseudonucleus thuja acid of at least a following formula

X-Y-Q

Wherein X is the backbone monomers unit that can insert nucleic acid or nucleic acid analog main chain,

42. the oligonucleotide of claim 41 or oligonucleotide analogs, wherein the total length of Q and Y at 8  in the scope of 13 .

43. the oligonucleotide of claim 42 or oligonucleotide analogs, condition are when intercalator is pyrene, the total length of Q and Y at 9  in the scope of 11 .

44. each oligonucleotide or oligonucleotide analogs of claim 41-43, wherein intercalator pseudonucleus thuja acid such as claim 1 to 25 each define.

45. each oligonucleotide or oligonucleotide analogs of claim 41-44, wherein oligonucleotide or oligonucleotide analogs synthetic such as claim 40 definition.

46. each oligonucleotide or oligonucleotide analogs of claim 41-45, wherein oligonucleotide or oligonucleotide analogs comprise two intercalator pseudonucleus thuja acids.

47. each oligonucleotide or oligonucleotide analogs of claim 41-46, wherein oligonucleotide or oligonucleotide analogs comprise that one or more is selected from DNA, RNA, PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2 '-NH)-TNA, (3 '-NH)-TNA, α-L-ribo-LNA, α-L-wood sugar-LNA, β-D-wood sugar-LNA, α-D-ribo-LNA, [3.2.1]-LNA, two ring-DNA, 6-amino-two ring-DNA, 5-shows-two ring-DNA, α-two ring-DNA, three ring-DNA, two ring [4.3.0]-DNA, two ring [3.2.1]-DNA, two ring [4.3.0] acid amides-DNA, β-D-ribopyranose base-NA, α-L-pyrans lysol glycosyl-NA, 2 '-R-RNA, 2 '-OR-RNA, α-L-RNA, the subunit of β-D-RNA.

48. each oligonucleotide or oligonucleotide analogs of claim 41-47, wherein the fluorescent characteristic of intercalator pseudonucleus thuja acid changes after under the predetermined stringent condition described oligonucleotide or oligonucleotide analogs being hybridized to corresponding nucleic acids or nucleic acid analog.

49. each oligonucleotide or oligonucleotide analogs of claim 41-48, wherein the melting temperature (Tm) of the crossbred of being made up of described oligonucleotide and homology complementary DNA (DNA crossbred) is significantly higher than the melting temperature (Tm) of the crossbred (corresponding D NA crossbred) of the oligonucleotide of not being with the pseudonucleus thuja acid and homology complementary DNA, and the oligonucleotide of described shortage intercalator pseudonucleus thuja acid comprises the nucleotide sequence identical with described oligonucleotide analogs.

50. the oligonucleotide analogs of claim 49, wherein the melting temperature (Tm) of DNA crossbred is higher at least 3 ℃ than the melting temperature (Tm) of corresponding D NA crossbred.

51. each oligonucleotide or oligonucleotide analogs of claim 41-48, wherein the melting temperature (Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and homology complementary DNA (DNA heterozygote) is apparently higher than the melting temperature (Tm) of the heterozygote of being made up of described oligonucleotide or oligonucleotide analogs and a homology complementary RNA (RNA crossbred).

52. the oligonucleotide of claim 51 or oligonucleotide analogs, wherein the melting temperature (Tm) of DNA crossbred is higher at least 5 ℃ than the melting temperature (Tm) of RNA crossbred.

53. the oligonucleotide of claim 51 or oligonucleotide analogs, wherein the melting temperature (Tm) of DNA crossbred is higher at least 10 ℃ than the melting temperature (Tm) of RNA crossbred.

54. the method for separation sequence specific DNA from the mixture that comprises nucleic acid, described method comprises the steps:

A) provide a mixture that comprises nucleic acid; And

B) provide one or more different oligonucleotide or oligonucleotide analogs, wherein the melting temperature (Tm) by described oligonucleotide or an oligonucleotide analogs and a corresponding DNA (the crossbred DNA crossbred of forming) is significantly higher than by the melting temperature (Tm) that comprises the crossbred (RNA crossbred) that described oligonucleotide or oligonucleotide analogs and a corresponding RNA form, and wherein said oligonucleotide or oligonucleotide analogs can with described sequence specific DNA hybridization; And

C) described mixture and described oligonucleotide or oligonucleotide analogs are incubated under the condition that allows hybridization between described oligonucleotide or the similar and described sequence specific DNA of oligonucleotide (DNA crossbred); And

Obtain isolating sequence specific DNA and the isolating remaining mixture that comprises nucleic acid thus.

55. the method for claim 54 is substantially free of sequence specific DNA comprising the remaining mixture of nucleic acid.

56. the method for claim 54, its amplifying nucleic acid are RNA and DNA.

57. the method for claim 54, wherein oligonucleotide or oligonucleotide analogs are selected from the oligonucleotide of each definition of claim 41-53.

58. the method for claim 54 wherein provides from 1 to 10,000 kind of different oligonucleotide and/or oligonucleotide analogs.

59. the method for claim 54, wherein oligonucleotide or oligonucleotide analogs be by 5 to 10, such as 10 to 15, and for example 15 to 20, such as 20 to 30, for example 30 to 100 Nucleotide and/or nucleotide analog and/or intercalator pseudonucleus thuja acid are formed.

60. the method for claim 54, wherein oligonucleotide is attached on the solid support.

61. the method for claim 60, wherein solid support is an activating surface.

62. the method for claim 60, wherein solid support is selected from magnetic bead, sepharose 4B, sepharose pearl, glass, plastic surface, heavy metal and chip surface.

63. a method that detects sequence specific DNA (target DNA) in the mixture that comprises nucleic acid and/or nucleic acid analog, described method comprises the steps:

A) provide nucleic acid mixture; And

B) provide one or more different oligonucleotide or oligonucleotide analogs, wherein the melting temperature (Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and corresponding D NA (DNA crossbred) is significantly higher than the melting temperature (Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and corresponding RNA (RNA crossbred), and wherein said oligonucleotide or oligonucleotide analogs are complementary to described sequence specific DNA (target DNA) basically; And

C) described mixture and described oligonucleotide or oligonucleotide analogs are incubated under hybridization conditions; And

D) detect oligonucleotide or the oligonucleotide analogs that hybridizes to sequence specific DNA; With

Detect described sequence specific DNA thus.

64. the method for claim 63, wherein mixture comprises sequence specific DNA and the RNA with similar sequence.

65. the method for claim 63, wherein oligonucleotide or oligonucleotide analogs are selected from the oligonucleotide or the oligonucleotide analogs of each definition of claim 41 to 53.

66. the method for claim 63, wherein said method comprise the step that at least a oligonucleotide or oligonucleotide analogs is fixed to solid support.

67. the method for claim 666, wherein solid support is selected from chip array upholder and microtiter plate.

68. detect the method for sequence-specific RNA from the mixture that comprises nucleic acid and/or nucleic acid analog, described method comprises the steps:

A., nucleic acid mixture is provided; And

B., one or more different oligonucleotide or oligonucleotide analogs are provided, wherein the melting temperature (Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and corresponding DNA (DNA crossbred) is significantly higher than the melting temperature (Tm) of the crossbred of being made up of described oligonucleotide or oligonucleotide analogs and corresponding RNA (RNA crossbred), and wherein said oligonucleotide or oligonucleotide analogs are complementary to described sequence-specific RNA basically; And

C. provide one comprise detectable label and one can with the probe of the nucleotide sequence of described sequence-specific RNA hybridization; And

D. described mixture and described oligonucleotide or oligonucleotide analogs are incubated under the condition of hybridization, thereby seal arbitrary sequence specific DNA; And

E. described mixture and described probe are incubated under hybridization conditions; And

F. detect described detectable label; And

Detect described sequence-specific RNA thus.

69. the method for claim 68, wherein oligonucleotide or oligonucleotide analogs are the oligonucleotide or the oligonucleotide analogs of each definition of claim 41 to 53.

70. the method for claim 68, wherein mixture comprises DNA and RNA.

71. the method for claim 68, wherein mixture is included in the cell.

72. comprise that the oligonucleotide or the oligonucleotide analogs of first sequence and second sequence are right, described first sequence is oligonucleotide and/or the oligonucleotide analogs that comprises at least a intercalator pseudonucleus thuja acid, and second sequence can hybridize to described first sequence, and each defines comprising the oligonucleotide of first sequence or oligonucleotide analogs such as claim 41-53.

73. the oligonucleotide or the oligonucleotide analogs of claim 72 are right, wherein intercalator pseudonucleus thuja acid such as claim 1-25 each define.

74. the oligonucleotide or the oligonucleotide analogs of claim 72 or 73 are right, wherein second sequence comprises at least one intercalator pseudonucleus thuja acid.

75. each oligonucleotide or oligonucleotide analogs of claim 72-74 is right, wherein oligonucleotide or oligonucleotide analogs are selected from RNA, 2 '-O-methyl RNA, LNA, PNA, HNA, MNA and ANA.

76. each oligonucleotide or oligonucleotide analogs of claim 72-76 is right, wherein oligonucleotide or oligonucleotide analogs comprise at least two intercalator pseudonucleus thuja acids, and it is closely approaching when described sequence hybridization.

77. one comprises the oligonucleotide and/or the oligonucleotide analogs of the insertion pseudonucleus thuja acid of at least one each definition of claim 41-53, the intercalator of wherein said insertion pseudonucleus thuja acid is that a fluorophor and wherein said oligonucleotide or oligonucleotide analogs can hybridize to corresponding D NA, and wherein said hybridization causes the minimizing of described oligonucleotide analogs fluorescence intensity.

78. a method that suppresses DNAse and/or RNAse comprises to RNAses and/or DNAses increasing at least a oligonucleotide and/or comprising the oligonucleotide of at least one pseudonucleus thuja acid.

79. the method for claim 74, wherein at least one oligonucleotide and/or oligonucleotide such as claim 41-53 each define.

80. regulate the method that one or more specific genes are transcribed for one kind, described method comprises the steps:

A) provide a re-reading system;

B) provide at least a oligonucleotide analogs as claim 41-53 definition,

D) allow oligonucleotide and/or oligonucleotide analogs and described one or more genes and/or its to regulate the complementary strand hybridization that sequence or gene and/or its are regulated sequence; And

Thereby regulate described gene transcription.