WO1995005391A1 - Applications de n-nucleosides fluorescents et d'analogues structurels fluorescent de n-nucleosides - Google Patents

Applications de n-nucleosides fluorescents et d'analogues structurels fluorescent de n-nucleosides Download PDF

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WO1995005391A1
WO1995005391A1 PCT/US1994/009316 US9409316W WO9505391A1 WO 1995005391 A1 WO1995005391 A1 WO 1995005391A1 US 9409316 W US9409316 W US 9409316W WO 9505391 A1 WO9505391 A1 WO 9505391A1
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fluorescent
dna
sequence
nucleic acid
probe
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PCT/US1994/009316
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Michael J. Conrad
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Chromagen, Inc.
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Priority to JP7507174A priority Critical patent/JPH09505556A/ja
Priority to EP94927183A priority patent/EP0669928A1/fr
Publication of WO1995005391A1 publication Critical patent/WO1995005391A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical

Definitions

  • N-nucleosides which predominate in the composition of DNA and RNA from all sources have the structures shown in Figure 1 wherein R 6 is H for inosine and NH 2 for guanosine, R g is H for uridine and CH 3 for thymidine. Furthermore, R 12 ,
  • the six commonly occurring nucleotides do not absorb light at wavelengths >290 nm and are effectively non-fluorescent under physiological conditions.
  • Derivatives of the commonly occurring N-nucleotides for a variety of synthetic, diagnostic, and therapeutic purposes are common, including substitutions on both the heterocyclic base and the furanose ring.
  • R v R 2 , and R 3 can be H, OH, alkyl, acyl, amide, thioether, or disulfide);
  • R 5 is H or part of an etheno linkage with R 4 ;
  • Rg is hydrogen, methyl, bromine, fluorine, or iodine, or an alkyl or aromatic substituent, or an optional linking moiety including an amide, thioether, or disulfide linkage or a combination thereof such as R 1 - Cti 2 )_-R 2 , or R 1 -R 2 -(CH 2 ) ⁇ -R 3 -, where x is an integer in the range of 1 and 25 inclusive;
  • R 10 is hydrogen, or an acid-sensitive base stable blocking group, or a phosphorous derivative,
  • R 12 is
  • N and C in the N-nucleosides and C-nucleosides designate the atom at which the glycosidic covalent bond connects the sugar and the heterocyclic base.
  • the bases are either adenine, guanine, cytosine, inosine, uracil, or thymine.
  • the bases are attached to a furanose sugar, a general structure of which is shown in Figure 3.
  • the sugar substituents for the fluorescent analogs share the same numbering system for all R groups, but the numbering system for some of the heterocycle analogs may differ.
  • Nucleotide sequences are commonly utilized in a variety of applications including diagnostic and therapeutic probes which hybridize target DNA and RNA and amplification of target sequences. It is often necessary, or useful, to label nucleotide sequences.
  • Hybridization of specific DNA or RNA sequences typically involves annealing oligonucleotides of lengths which range from as little as 5 bases to more than 10,000 bases (10 kb).
  • the majority of oligonucleotide probes currently in research use are radioactively labeled; however, because of (a) the short half lives of the isotopes in common usage, (b) the safety requirements, and (c) the costs of handling and disposal of radioactive probes, convenient and sensitive non-isotopic methods of detection are required for hybridization diagnostic methods to achieve widespread acceptance and application.
  • Non-isotopic methods of labeling oligonucleotide probes In general, all of the non-isotopic methods of detecting hybridization probes that are currently available depend on some type of derivatization of the nucleotides to allow for detection, whether through antibody binding, or enzymatic processing, or through the fluorescence or chemiluminescence of an attached "reporter" molecule. In most cases, oligonucleotides have been derivatized to incorporate single or multiple molecules of the same reporter group, generally at specific cyclic or exocyclic positions.
  • DNA probes have been amino modified and subsequently derivatized to carry a hapten such as 2,4-dinitrophenol (DNP) to which enzyme- conjugated anti-hapten antibodies bind which subsequently can be processed using a colorimetric substrate as a label (Keller et al [1988] Analytical Biochemistry 170:441-450).
  • DNP 2,4-dinitrophenol
  • Patent No.4,910,300 covering pyrimidine derivatives on which the 6-amino group at C 4 had been modified.
  • 3' and 5' amino modifying phosphoramidites have been widely used in chemical synthesis or derivatized oligonucleotides and are commercially available.
  • nucleotide triphosphate derivatives are effectively incorporated into double stranded DNA by standard techniques of "nick translation.” Once in an oligonucleotide, the residue may be bound by avidin, streptavidin, or anti-biotin antibody which can then be used for detection by fluorescence, chemiluminescence, or enzymatic processing.
  • 11-digoxigenin-ddUTP labeling The enzyme, terminal transferase, has been used to add a single digoxigenin-11-dideoxyUTP to the 3' end of oligonucleotides. Following hybridization to target nucleic acids, DIG-ddUTP labeled hybridization probes were detected using anti-DIG antibody conjugate. (6) AAIF. Immunofluorescent detection can be done using monoclonal Fab' fragments which are specific for RNADNA hybrids in which the probe has been derivatized with, e.g., biotin-11-UTP (Bobo et al [1990] /. Clin. Microbiol 28:1968-1973; Viscidi et al [1986] J. Clin. Microbiol 23:311-317).
  • Rhodamine derivatized probes are commercially available which hybridize to specific target DNAs and which can be detected using a flow cytometer or a microscope. Numerous authors have reported coupling fluorophores to chemically synthesized oligonucleotides which carried a 5' or 3' terminal amino or thiol group (Brumbaugh et al [1988] Nucleic Acids Res. 16:4937-4956). (9) Direct enzyme labeling. Chemical coupling of an enzyme directly to a chemically synthesized probe has been used for direct detection through substrate processing.
  • Urdea et al described an oligonucleotide sandwich assay in which multiple DNA probe hybridizations were used to bind target DNA to a solid phase after which it was further labeled with additional, alkaline phosphatase-derivatized hybridization probes (Urdea et al [1989] Clin. Chem. 35:1571-1575).
  • Acridinium ester labeling A single phenyl ester of methyl acridinium is attached at a central position on an RNA or DNA probe. Hydrolysis of the ester releases an acridone, C0 2 , and light. Because the ester on unhybridized probes hydrolyzes more quickly than the ester on probes which have hybridized to target RNA or DNA, the chemiluminescence of the hybridized probes can be distinguished from that of free probes and is used in a "hybridization protection assay" (Weeks et al [1983] Clin. Chem. 29:1474-1479). D. Derivatizations of the furanose ring CF). Methods for derivatization of the furanose ring (R ⁇ through R 14 in Figure 3) and at the phosphodiester backbone of oligonucleotides (R 10 in Figure 3) have been reported.
  • Internucleotide linkage reporter groups R 10 sitel. Phosphoro-thioate esters have been used to provide a binding site for fluorophores such as monobromobimane
  • Chemically derivatized dNTPs are generally not cost-effective for use as stock deoxynucleotide triphosphates in PCR amplification, hence, labeling of amplified DNA is limited to (i) amplification using previously labeled primers, or (ii) annealing with labeled hybridization probes. Use of the former frequently results in false positives during amplification owing to (i) non-specific annealing of primers to non-target segments of DNA during amplification, or (ii) contamination by amplicons present in the laboratory environment which are residual from previous amplification experiments. Expense and technical difficulties in post- hybridization processing have largely limited the applications of labeled hybridization probes to research.
  • Formycin A (generally referred to as Formycin), the prototypical fluorescent nucleoside analog, was originally isolated as an antitumor antibiotic from the culture filtrates of Nocardia interforma (Hori et al [1966] J. Antibiotics, Ser. A 17:96-99) and its structure identified as 7-amino-3-b-D-ribafuranosyl (lH-pyrazolo-[4,3d] pyrimidine)) ( Figures 5 and 6).
  • This antibiotic which has also been isolated from culture broths oi Streptomyces lavendulae (Aizawa et al [1965] Agr. Biol Chem. 29:375-376), and Streptomyces gummaensis (Japanese Patent No. 10,928, issued in 1967 to Nippon Kayaku Co.,
  • Formycin, formycin B, and oxoformycin B are pyrazolopyrimidine nucleosides and are structural analogs of adenosine, inosine, and hypoxanthine, respectively; a pyrazopyrimidine structural analog of guanosine obtained from natural sources has not been reported in the literature. A thorough review of the biosynthesis of these compounds is available in Ochi et al (1974) /. Antibiotics xxiv:909-916.
  • nucleoside analogs Physical properties of the nucleoside analogs. Because several of the C-nucleosides were known to be active as antibiotic, antiviral, or anti-tumor compounds, their chemical derivatization and physical properties have been extensively studied and compared to the structures and syntheses of the N-nucleosides commonly found in DNA and RNA In the late 1960s, several structural analogs of the six commonly occurring N-nucleosides were found to be fluorescent under physiological conditions; fluorescence in the analogs results from a molecular rigidity of the heterocycle structure itself, not all the structural analogs of a given type, e.g., the C-nucleosides, are fluorescent, nor is fluorescence an exclusive or inherent property of any particular class of structural analogs.
  • the subject invention pertains to nucleoside analogs which are fluorescent. These fluorescent nucleoside analogs are useful as monomers in synthesizing and labelling nucleotide sequences.
  • the invention further pertains to the use of these fluorescent nucleotides which can be substituted for naturally occurring nucleosides in the synthesis of oligonucleotide probes. When used as hybridization probes, the fluorescence of such oligonucleotides can be used as a diagnostic tool to detect and identify specific genetic sequences.
  • This methodology is distinct from other non-radioactive methods of probe detection in that it does not utilize nucleotides which have been coupled to enzymes or other reactive proteins and does not require post-hybridization processing for the detection of hybridization.
  • enzymatic methods are provided for making nucleic acid probes which are complementary to, and will bind to, only the sense or only the anti-sense, but not both, strands of a DNA duplex (asymmetric synthesis). It is an important aspect of the invention that asymmetric synthesis is the necessary condition for creating rapid and quantitative nucleic acid probe tests, assays, diagnostics, and therapeutics.
  • a significant aspect of asymmetric synthesis is its dependence on the asymmetric use of promoters, primers, or linker modified primers to direct the synthesis or isolation of oligonucleotides or oligomers using only one of the two strands of a duplex as the template.
  • asymmetric synthesis makes possible the directed use of multiple different templates for concurrent synthesis of a "cocktail" of asymmetric probes which can hybridize concurrently to independent and unique target sites on a single piece of nucleic acid, genomic DNA, or chromosome. It is an important aspect of the invention of probe "cocktails" that if multiple copies of the same target sequence are present on a single genome, such as the multiple copies of the tandem repeat intergenic sequences disclosed in Example 7, a single asymmetric probe template can be used to create a "cocktail” which will bind to many targets on a single genome which are identical in sequence but widely distributed in locus on the genome.
  • fluorescent structural analogs of the commonly occurring nucleosides and their derivatives useful in the synthesis, labeling, and detection of oligonucleotides having the structural formulae of Figures 5 through 11.
  • the commonly occurring nucleosides characteristically form hydrogen bonds in a specific donor/acceptor relationship, designated Watson-Crick base pairing as shown in Figure 4.
  • specific fluorescent nucleoside analogs capable of reproducing the pattern of Watson-Crick hydrogen bond formation analogous to that of a particular commonly occurring nucleoside are provided, as indicated for, e.g., A:T and formycin:T in Figure 4 by the donor/acceptor patterns.
  • methods of making and derivatizing the fluorescent structural analogs of the commonly occurring nucleosides including the steps of derivatizing the R 10 , R 12 , and R 14 moieties to be (i) reactive in DNA or RNA synthesis, and/or (ii) reactive in Resonance Energy Transfer of the fluorescence from the structural analogs.
  • methods of synthesizing and using polynucleotide probes are provided using one or more of the fluorescent structural analogs and/or their derivatized forms.
  • Such probes can be used to screen a sample containing a plurality of single stranded or double stranded polynucleotide chains and will label, detect, and identify the desired sequence, if present, by hybridization.
  • the fluorescent oligonucleotide probes can be used with "solution hybridization" methods as depicted in Figures 12 through 18.
  • the present invention comprises inherently fluorescent nucleosides which can be used to label, modify, or identify oligonucleotides made therefrom, the uses of such inherently fluorescent oligonucleotides as hybridization probes, and methods for detecting nucleotide sequences.
  • An important aspect of the invention is the stable fluorescence emission of the fluorophores and the use of time-resolved spectroscopy or photon counting to detect and to quantify the amount of a fluorophore present in a sample.
  • Figure 1 shows the six commonly-occurring N-nucleosides which predominate in DNA and RNA
  • Figure 2 shows the general structures of the commonly-occurring N-nucleosides and their derivatization sites, R..
  • Figure 3 shows the general structure of the furanose ring of both the purine and pyrimidine nucleosides and the common sites, R. for derivatization.
  • Figure 4 shows Watson-Crick base pairing between the normally occurring N-nucleotides A:T and G:C and base pairing between formycin:T, formycimU, 2,6-diaminopurine:T, and 5- amino-formycin B:C.
  • Figure 5 shows structural analogs of the commonly-occurring N-nucleosides derived from biological sources.
  • Figure 8 shows the pyrazolo [l,5a]-l,3,5-triazine nucleoside analogs.
  • Figure 9 shows the azapyrimidine and azapurine nucleoside analogs.
  • Figure 10 shows the deazapyrimidine and deazapurine nucleoside analogs.
  • Figures 11A-11B shows examples of some fluorescent structural analogs which are (11A) non-H-binding, and (11B) fluorescence resonance energy transfer (FRET) analogs.
  • Figure 12 is a diagram of symmetric RNA synthesis using FTP or ATP.
  • Figure 13 is a diagram of promoter directed asymmetric RNA probe synthesis using viral promoters and viral RNA polymerases.
  • Figure 14 is a diagram showing an example of the method for one-step labeling of ssDNA inserted at the EcoRI site of pUC/M13 plasmid vectors and using dF 105 .
  • Figure 15 is a diagram showing the necessity of using asymmetric DNA or RNA probes for rapid and quantitative hybridization of the probe to target DNA As shown, asymmetric probes provide significant increases in hybridization efficiencies when compared with symmetric probes.
  • Figure 16 is a diagram showing the conversion of the ribonucloetide analog, formycin A, to its 2'-deoxy triphosphate or phosphoramidite forms.
  • Figure 17 is a diagram of detection of a target DNA sequence in genomic DNA hybridization with fluorescent probes.
  • Figure 18 is a diagram of detection of an amplified DNA segment by solution hybridization of a fluorescent probe.
  • Figure 19 shows a flow chart diagramming the separation scheme used to separate reaction products from unreacted reagents following the enzymatic substitution reaction of FTP for ATP in RNA probes.
  • Figure 20 shows a schematic of the mechanism for increasing detection sensitivity by the use of a probe "cocktail" which contains multiple probes of different sequences.
  • Figures 21A-21F-1 show specific fluorescent nucleoside analogs which have been identified and characterized as to their class, structure, chemical name, absorbance spectra, emission spectra, and methods of synthesis.
  • Figure 22 shows the 5' universal end label comprising four distinct functional groups which include: region A, a non-base-pairing homopolymer from 1 to about 50 fluorescent nucleoside analogs long; region B, an optional non-nucleoside phosphodiester "tether" connecting region A to region C; region C, an enzymatic synthesis primer, complementary to a promoter in the target sequence; and region D, a nucleotide chain from about 40 to about 20,000 nucleotides in length complementary to the target nucleotide sequence. Regions A, B, and C which are typically 20 to 60 bases in length can be chemically synthesized. Region D can preferably be enzymatically synthesized. The synthesis of the 5' universal end label is described in Example 8.
  • Figure 23 shows a schematic representation of the method for synthesizing a highly flourescent 5' labeled probe.
  • the method comprises the following steps: (1) restricting, with a specific restriction enzyme, a sequence having a known promoter site and known restriction site downstream from the known promoter site, (2) inserting a unique target sequence at the restriction site, (3) hybridizing a fluorescent nucleoside analog probe comprising a sequence complementary to the promoter of the inserted target sequence, and (4) extending the probe sequence from the hybridized promoter region, using a nucleic acid polymerase, to synthesize a specific probe complementary to the inserted target sequence.
  • Figure 24 shows the normalized spectrum profiles compared for F 185 (ethenoadenosine) and F 105 (formycin). The spectra were determined between 250-405 nm for the compounds, using a 2.5 nm slit.
  • Figure 25 shows a method for increasing sensitivity of detection or differential labeling using multiple copies of a 5' universal end label. Shown is an example of a target sequence, using of the probe is the 5' universal end label on each of a plurality of probe sequences where each of the probe sequences is complementary to a different fragment or segment of the target sequence.
  • Figure 26 shows the sequencing application of 5' universal end label.
  • the unique probe (which can be produced by the method shown in Figure 24) can be employed using DNA polymerase to produce a plurality of dideoxy fragments having different lengths.
  • Figure 27 shows the Sustained Signal Amplification procedure.
  • the example in the figure employs the genome from the Hepatitis B virion having two unequal lengths of DNA forming its double-stranded genome.
  • the method shows the steps: (1) extension of the shorter DNA strand using nucleotides or phosphorylated nucleoside analogs; (2) the separation of the two " strands and the addition of two primers, A and B; (3) extension of the DNA strand to which the A primer is hybridized using reverse transcriptase; (4) utilization of the synthesized double-stranded sequence in an amplification cycle which comprises (a) production of antisense RNA template from the synthesized DNA using an RNA polymerase, (b) synthesis of DNA from the RNA template using primer B and a nucleic acid polymerase, and (c) synthesis of double stranded DNA (DNA replicate) from the B primer-DNA
  • the replicate DNA can then repeat the signal amplification cycle (step (4)).
  • SEQ ID NO. 1 is a synthetic oligonucleotide according to the subject invention.
  • SEQ ED NO. 2 is a synthetic oligonucleotide and the complement of SEQ ID NO. 1.
  • SEQ ID NO. 3 is a synthetic oligonucleotide and a fluorescent analog of SEQ ID NO.2.
  • novel fluorescent nucleoside analogs and methods of use of the fluorescent nucleosides in, for example, nucleic acid probes and diagnostic kits pertains to the use of inherently fluorescent nucleoside analogs in the chemical and enzymatic synthesis of DNA hybridization probes including solid phase synthesis, template directed enzymatic polymerization and amplification using polymerase chain reaction methods.
  • Another embodiment relates to the use of autofluorescent DNA hybridization probes in the identification of specific DNA sequences, e.g., gene mapping and the detection and diagnosis of infectious and genetic diseases.
  • the subject invention pertains to nucleoside analogs which are fluorescent and which can be substituted for naturally occurring nucleosides in the synthesis of oligonucleotide probes.
  • the fluorescence of such oligonucleotides can be used in a variety of procedures to detect and identify specific genetic sequences. This methodology is distinct from other non-radioactive methods of probe detection in that it does not utilize nucleotides which have been coupled to enzymes or other reactive proteins.
  • described herein are applications of inherently fluorescent nucleoside analogs in developing hybridization techniques for routine, automatable clinical diagnosis.
  • the fluorescent analogs of the subject invention are of three general types: (A) C- nucleoside analogs; (B) N-nucleoside analogs; and (C) N-azanucleotide and N-deazanucleotide analogs. All of these compounds have three features in common: 1) they are structural analogs of the common nucleosides capable of replacing naturally occurring nucleosides in enzymatic or chemical synthesis of oligonucleotides; 2) they are naturally fluorescent when excited by light of the appropriate wavelength(s) and do not require additional chemical or enzymatic processes for their detection; and 3) they are spectrally distinct from the nucleosides commonly encountered in naturally occurring DNA At least 125 specific compounds of the subject invention have been identified. These compounds, which have been characterized according to their class, structure, chemical name, absorbance spectra, emission spectra, and method of synthesis, are tabulated as shown in Figures 21A-21F-1.
  • Communication Occurring Nucleosides are the six monomeric N-nucleotides shown in Figure 1, which predominate in naturally occurring DNA and RNA, enter into classical Watson-Crick base pairing, and are effectively non-fluorescent under physiological conditions.
  • the respective one-letter symbols in sequence shorthand are A, C, G, T, U, and I for adenosine, cytidine, guanidine, thymidine, uridine, and inosine, respectively.
  • Structural Analogs of the commonly occurring nucleosides are structurally related molecules that mimic the normal purine or pyrimidine bases in that their structures (the kinds of atoms and their arrangement) are similar to the commonly occurring bases, but may have certain modifications or substitutions which do not affect basic biological activity or biochemical functions.
  • Such base analogs include, but are not limited to, imidazole and its 2,4- and/or 5- substituted derivatives; indole and its 2-, 3-, 4-, 5-, 6-, and/or 7-substituted derivatives; benzimidazole and its 3-, 4-, and/or 5-substituted derivatives; indazole and its 3-, 4-, 5-, 6-, and/or 7- substituted derivatives; pyrazole and its 3-, 4-, and/or 5-substituted derivatives; triazole and its 4- and/or 5-substituted derivatives; tetrazole and its 5-substituted derivatives; benzotriazole and its 4-, 5-, 6-, and/or 7-substituted derivatives; 8-azaadenine and its substituted derivatives; 6- azathymine and its substituted derivatives; 6-azauracil and its substituted derivatives; 5-azacytosine and its substituted derivatives; 8-azahypoxanthine and its substituted derivative
  • Base analogs can also be any of the C-nucleosides such as are shown in Figures 4 and 5 in which the normal C-N bond between the base and the furanose ring is replaced by a C-C bond; such bases include, but are not limited to, uracil, as in the C-nucleoside pseudouridine; 1- methyluracil;l,3-dimethyluracil;5(4)-carbometh ⁇ 3ty-l,2,3-triazole;5(4)-carboxamido-l,2,3-triazole; 3(5)-carboxymethylpyrazole; 3(5)-carbomethoxypyrazole; 5-carboethoxy-l-methylpyrazole; maleimide (in the C-nucleoside showdomycin); and 3(4)-carboxamido-4(3)-hydroxypyrazole (in the C-nucleoside pyrazomycin); and any of the other analogs listed or inferred in Figures 5 through 11; or their protected derivatives.
  • Fluorophore refers to a substance or portion thereof which is capable of emitting fluorescence in a detectable range.
  • this fluorescence typically occurs at wavelengths in the near ultraviolet (>300 nm) through the visible wavelengths.
  • fluorescence will occur at wavelengths between 300 nm and 700 nm and most preferably in the visible wavelengths between 300 nm and 500 nm.
  • “Fluorescent Structural Analogs” are synthetic or biochemically derived monomeric structural analogs of the six commonly occurring N-nucleosides ( Figure 1), such as are depicted in Figures 5 through 11, which may or may not be capable of classical Watson-Crick base pairing depending upon the monomeric structure and/or oligonucleotide in which they are used, but which are spectrally unique and distinct from the commonly occurring nucleosides in their capacities for selective excitation and emission under physiological conditions.
  • "Derivatized" nucleoside analogs are fluorescent structural analogs in which reactive or protective functional groups are bound, covalently or otherwise, at the R 4 through Rg positions of the heterocycle and/or the R 10 (5'), the R 12 (3'), and R 14 (2') positions of the glycosidic moiety. Derivatives at the 2' glycosidic position may include fluorescence resonance energy transfer (FRET) acceptors or donors which enhance or accept and re-emit at longer wavelengths the inherent fluorescence emission of the fluorescent structural analog itself.
  • FRET fluorescence resonance energy transfer
  • a “polynucleotide,” “oligonucleotide,” or “oligomer” is a nucleotide chain structure containing at least two commonly occurring nucleotides or fluorescent structural analogs.
  • the “fluorescent oligonucleotide probe” or “fluorescent hybridization probe” provided herein is a nucleotide chain structure, as above, containing at least two monomers, at least one of which is fluorescent.
  • Hybridization is the pairwise annealing through Watson-Crick base pairing of two complementary, single-stranded molecules (see Figure 4), which may be DNADNA, DNARNA or RNARNA, and in which the two strands may come from different sources.
  • the annealing is specific (i) for complementary base pairs in which the hydrogen bond donors and acceptors are oriented as in Figure 4, and (ii) for the complementary genetic sequence of the specific gene, target DNA, or target RNA (hereinafter “target DNA/RNA”) to which the probe is to be hybridized.
  • target DNA/RNA complementary genetic sequence of the specific gene, target DNA, or target RNA
  • DNA RNA Melting Temperature and “Tm” refer to the temperature at which the hydrogen bonds between hybridized strands of DNA or RNA are disrupted and the strands disassociate into single strands, thereby disrupting the structure of the duplex or hybrid.
  • Analogous fluorescent sequence refers to the nucleoside sequence of a polynucleotide which has been synthesized by any of the enzymatic or chemical methods described in the present invention, but in which fluorescent nucleoside analogs have been explicitly substituted for particular commonly occurring nucleosides, e.g., the substitution of formycin A-5'-triphosphate (FTP) for adenosine-5 '-triphosphate (ATP), when using RNA polymerase to produce RNA probes complementary to a prescribed DNA template.
  • FTP formycin A-5'-triphosphate
  • ATP adenosine-5 '-triphosphate
  • the fluorescent nucleoside analog has been substituted in the oligonucleotide chain at some or all positions in which the corresponding commonly occurring nucleotide would have occurred in the sequence as dictated by, e.g., the template, in the case of enzymatic synthesis. Similar programmed substitutions can be made using 3'-0-phosphoramidites of the individual fluorescent analogs during standard phosphotriester synthesis.
  • the complementary sequence of the Chlamydia tracheomatis MOMP gene, or its fluorescent analogous sequence can be synthesized enzymatically using dATP or dFTP, respectively, in the presence of DNA polymerase, dCTP, dTTP, and dGTP:
  • FRET acceptor or “Fluorescence Resonance Energy Transfer acceptor” refers to a substance, substituent, chromophore, or fluorophore, e.g., a dansyl, naphthyl, anthryl, pyrenyl, methylumbelliferone, or coumarin moiety, which is capable of absorbing emitted light from fluorescent structural analog donors and re-emitting that energy at other, longer wavelengths.
  • such secondary fluorophores may be selectively excited as a second label, or may be used as a fluorescence acceptor to broaden and enhance the primaiy fluorescence of the structural analog energy donor.
  • 2-amino purine ribonucleoside, and 2,6-diamino ribonucleoside all of which can (i) form the same or related base-pairing hydrogen bonds as adenosine, and (ii) substitute specifically for adenosine in Watson-Crick base pairing as well as in a wide variety of enzymatic reactions including nucleic acid replication, ligation, and phosphoiylation, are used as representatives of the set of fluorescent nucleosides and nucleoside analogs ( Figure 4).
  • Related properties and parallel claims obtain in the present invention for all other fluorescent analogs of guanosine, cytidine, thymidine, uridine, inosine, and their derivatives.
  • Furanose moieties common to the fluorescent nucleoside analogs The numbering of the sugar carbon atoms in furanose is 1' to 5' as indicated in Figure 2; thus the base, B, is connected to CI of the sugar.
  • the furanose moiety of any fluorescent heterocycle claimed in this invention has, in common with all other analogs, the set F, of glycosides and substituted glycosides, as follows: substitutions can be made, in principle, at any of the 5 sugar carbons; the subset F is defined by derivatives and/or substitutions at positions R 10 , R ⁇ , R 12 , R 13 , and R 14 , which (i) are apparent to one skilled in the art, and (ii) are the furanosyl derivatives of all the fluorescent nucleoside analogs claimed in the present invention.
  • R 12 and R 10 are either H, OH, OR,,,, or NHR k , wherein (a) R j protecting groups are typically lower aryl or alkyl ether, e.g., methyl, t-butyl, benzyl, o-nitrobenzyl, p-nitrobenzyl, o- nitrophenyl, or triphenylmethyl; or a lower alkyl or aryl ester such as acetyl, benzoyl, or p- nitrobenzoyl, or an alkyl; acetal such as tetrahydropyranyl; or a silyl ether, such as trimethylsilyl or t-butyl-dimethylsilyl; or a sulfonic acid ester such as p-toluenesulfonyl or methanesulfonyl; or halide such as bromine, fluorine, or iodine.
  • R j protecting groups
  • R 14 may be a FRET derivative including, but not limited to, such fluorophores as 7-[3-(chlorodimethylsilyl)propoxy]-4-methylcoumarin, 0-4-methylcoumarinyl-N-[3- triethoxysilyl)propylcarbamate, and N-3-triethoxysilylpropyl)dansylamide;
  • R ⁇ represents an appropriate protecting, substituting, or reactive linker group including 2' or 3'-amido, 2' or 3'- azido, 2',3'-unsaturated, and the subset of phosphorous derivatives involved in chemical or enzymatic syntheses of oligonucleotides having a phosphate ester, thiophosphate ester, or aminophosphate ester backbone;
  • R k is any common
  • the invention further includes novel phosphoramidites having the formula:
  • R 10 , R ⁇ , R 12 , R 13 are as defined for the set of glycosides, F, as above, and R 14 may be either H or OH.
  • R 16 lower alkyl, preferably lower alkyl such as methyl or isopropyl, or heterocyclic, such as morpholino, pyrrolidono, or 2,2,6,6-tetramethylpyrrolidono;
  • R 15 methyl, beta-cyanoethyl, p- nitrophenyl, o-chloronitrophenyl, or p-chlorophenyl.
  • All other R groups are as before including those identifying spacer or linker arms of from 1 to 25 carbon atoms in length.
  • the base moiety B in the phosphoramidite can be protected, which generally involves acylation or amidation of the exocyclic amino groups and includes, but is not limited to, acetyl, benzoyl, isobutiyl, succcinyl, phthaloyl, or p-anisoyl; such amidine groups include, but are not limited to, dimethylformamidine, di-n-butylformamidine, or dimethylacetamidine; if B is substituted with other reactive groups such as carboxyl, hydroxyl, or mercapto, these are appropriately protected as well.
  • the present invention encompasses the synthesis of oligonucleotides on a solid phase support, wherein the oligomer is reacted with the protected fluorescent nucleoside analog phosphoramidites as illustrated in Figures 5 through 11 and derivatized as in the structure, above. Additionally, the present invention includes the novel fluorescent oligonucleotides having included in their sequences at least one fluorescent nucleoside analog derivatized as the phosphoramidite in the structure, above. Moreover, it is yet again another aspect of the present invention to provide fluorescent oligonucleotides made by the reactions of the aforementioned fluorescent analog 3'-0-phosphoramidites which are bound to, or have been bound by, a solid support.
  • Formycin A is isolated as the ribonucleotide from the culture broths of Nocardia interforma.
  • the antibiotic is also isolated from culture broths of Streptomyces lavendulae and Streptomyces gummaensis, and is one of numerous microbial C-ribonucleoside analogs of the N-nucleosides commonly found in RNA from all sources.
  • the other naturally occurring C-ribonucleosides which have been isolated from microorganisms ( Figure 5) include formycin B, oxoformycin B, pseudouridine, showdowmycin, pyrazomycin, and minimycin.
  • Formycin A, formycin B, and oxoformycin B are C-nucleosides or pyrazolopyrimidine nucleosides of the class shown in Figure
  • C-nucleoside analogs of the pyrazolo-s-triazine class were prepared from amino pyrazole-C-nucleoside as originally described (Fox et al [1976] /. Heterocycl Chem. 13:175). Production of the deoxy. dideoxy. and phosphorylated forms of the fluorescent ribonucleoside analogs.
  • mono- and triphosphate forms of the nucleoside analogs can be prepared by enzymatic phosphorylation with, e.g., polynucleotide kinase using established procedures, or by chemical phosphorylation.
  • the 5'-monophosphates are prepared chemically by the POCl 2 (Smith and Khorana [1958] J. Am. Chem. Soc. 80:1141; Yoshikawa et al [1967] Tetrahedron Lett. 5095).
  • the corresponding triphosphates can be chemically synthesized according to the same authors or Michelson ([1964] Biochim. Biophys. Ada 91:1); or Hoard and Ott ([1965] /.
  • the present invention presents synthetic methods for the introduction of one or more of the fluorescent nucleoside analogs of the commonly occurring nucleotides into synthetic oligonucleotides.
  • the -cyanoethyl derivatives may be selectively inserted at any desired position in a chemically synthesized oligonucleotide to produce oligomers of prescribed sequences of 60 or more bases in length and carrying any predetermined number of fluorescent bases.
  • one aspect of the present invention involves the synthesis of 3'-0-phosphoramidites of the fluorescent nucleotides and of their fluorescent structural analogs, the use of amidites to synthesize highly fluorescent oligonucleotides having prescribed sequences and the uses of such oligonucleotides as amplification primers, fluorescent oligonucleotide "tags," and hybridization probes.
  • Fluorescent polyribonucleotides and polydeoxy-ribonucleotides of prescribed sequences can be synthesized enzymatically using DNA templates from a variety of sources including those prepared by chemical synthesis, cloning techniques, or obtained from genomic DNA Representative syntheses of RNA oligonucleotides using three such DNA templates, E. coli RNA polymerase, the rNTPs cytidine, uridine, and guanosine, together with the ribose triphosphate of either formycin A or adenosine, are illustrated in Figure 12.
  • RNA probe using a template bearing directional viral promoters, the viral RNA polymerases, the rNTPS cytidine, uridine, and guanosine together with the ribose triphosphate of either formycin A or adenosine, is illustrated in Figure 13.
  • Symmetric polydeoxyribonucleotides have been made by substituting 2'-deoxyformycin A-5 '-triphosphate (FTP) for deoxyadenosine-triphosphate (dATP) in standard DNA polyerase syntheses and in DNA amplifications using thermostable DNA polymerase enzymes and the polymerase chain reaction; the corresponding asymmetric syntheses have been achieved using the same reagents and procedures but with the following modifications: (i) syntheses using such DNA polymerase as Klenow fragment or modified T7 DNA polymerase incorporated into one strand of a duplex at the beginning of the sequence that was to be used as the template, and the corresponding primer was used to initiate all syntheses; (ii) primers complementary to only one strand of a template were used in amplification as is commonly described as asymmetric PCR; or (iii) paired primers in which one of each pair of primers was coupled to a linker such as biotin were used in standard DNA amplifications such as
  • Comparable syntheses can be made by other substitutions, including, e.g., the fluorescent N-nucleosides, 2-amino purine, and 2,6-amino purine (also substituted for adenosine-5'-triphosphate) and either of the fluorescent C-nucleoside triphospates of formycin B or 5-amino-formycin B (substituted for inosine triphosphate and guanosine-triphosphate, respectively) in either their ribose and deoxyribose forms.
  • substitutions including, e.g., the fluorescent N-nucleosides, 2-amino purine, and 2,6-amino purine (also substituted for adenosine-5'-triphosphate) and either of the fluorescent C-nucleoside triphospates of formycin B or 5-amino-formycin B (substituted for inosine triphosphate and guanosine-triphosphate, respectively) in either
  • RNA and DNA can be enzymatically labeled by several methods including, but not limited to, (i) 5' DNA end-labeling using both the forward phosphorylation reaction (Richardson, C.C.
  • Hybridization of the oligonucleotides with target DNA results in quenching of the fluorescence of the structural analogs in a fluorescent probe, which fluorescence is recovered upon denaturation of the hybrid, thereby proving that hybridization has occurred.
  • the self-hybridization of the synthetic oligonucleotide poly(rFrU), which is discussed at length, below, is representative of the results obtained in such experiments and is summarized in Table 1.
  • a preferred process according to the subject invention involves four basic steps. Initially the fluorescent structural analogs are chemically or biologicaUy synthesized and, where appropriate, further derivatized as required to synthesize a fluorescent oligonucleotide probe. Second, a DNA or RNA probe molecule complementary to a nucleic acid sample of interest is constructed to have fluorescent nucleoside analogs which can be (i) distributed randomly or at specific locations throughout its length, or (ii) placed as terminal labels as described below. Third, the nucleic acid sample is then separated from unreacted monomers and can then be characterized directly, used as an extrinsic, non-specific label for tagging specific hybridization probes, or used directly as a hybridization probe.
  • hybridization may take place on a solid phase to which either the target DNA/RNA or the fluorescent probe has been immobilized such as in Southern blot transfers, or "Dot-Blot” techniques, or it may occur in solution (herein, “solution hybridization”), after which probe/target hybrids are separated from unhybridized probes by simply washing or filtration. Finally, the fluorescence of the oligonucleotides hybridized to the target DNA/RNA is detected and quantified.
  • a preselected fluorescent nucleoside analog or mixture of fluorescent analogs is substituted specifically for one or more of the non-fluorescent commonly occurring nucleosides and is then incorporated into DNA or RNA oligonucleotides to create prescribed sequences.
  • the prescribed sequences may be chosen to be equivalent in their Watson-Crick base pairing to a nucleotide sequence constructed from normally occurring nucleotides and complementary to a given target DNA or RNA sequence; such fluorescent probes are said to be analogous to the complementary * sequence of the target DNA or RNA
  • the fluorescent probe may be synthesized by either enzymatic or chemical synthesis for subsequent applications such as (i) hybridization probes, (ii) amplimers for direct detection of amplifiable gene sequences complementary to a given set of primers, or (iii) as non-specific "universal" labels which can be attached to specific hybridization probes by, e.g., ligation.
  • Enzymatic syntheses include:
  • dAdenosine-5'-triphosphate dATP
  • dFTP dAdenosine-5'-triphosphate
  • fluorescent DNA oligonucleotides complementary to a specific DNA template can be synthesized (i) by using DNA fragments and E. coli DNA polymerase, or (ii) by constructing a recombinant plasmid containing the primer site for a specific primer such as the M13 forward primer immediately 5' to the desired DNA template sequence.
  • the DNA polymerase will synthesize a complementary DNA molecule using deoxyribonucleotides or other deoxyanalogs including, e.g., dFTP as a substitute for dATP, present in the reaction mixture;
  • an incorporation method which also produces a terminal concentration of fluorescent analogs involves the use of the "tailing" enzyme, terminal deoxynucleotide transferase, to add a homopolymer or "tail” of fluorescent deoxy analogs to the 3 ' end of DNA oligomers.
  • the yields obtained in the synthesis of homopolymers when substituting fluorescent analogs for the commonly occurring nucleosides is significantly less than the yield obtained in the synthesis of heteropolymers.
  • a single fluorescent nucleoside analog may be added to the 3' OH of any oligomer using the same enzyme but the dideoxy form of a fluorescent analog or a 2'-protected fluorescent analog, including the FRET protected analogs, in exactly the same manner in which, e.g., dideoxy ATP (cordecypin), is used.
  • a third alternative method of endlabeling hybridization probes utilizes the action of DNA ligase or RNA ligase, by which non-specific double or single stranded fluorescent oligonucleotides can be covalently coupled to either the 3' or 5' end of specific hybridization probes; the fluorescent oligonucleotides used in this fashion do not necessarily participate in the Watson-Crick base pairing which determines specificity of a probe, but may act solely as a generic or universal fluorescent "tag.”
  • the DNA probes are double stranded and must be denatured to single stranded form using either heat or alkali treatment prior to their use (d) an incorporation method, which can also be used as a standard method of production of fluorescent probes having a prescribed length and sequence, using standard methods of DNA amplification or replication and one of several available DNA polymerases, including but not limited to the thermostable DNA polymerases, e.g., Taq polymerase, modified T7 DNA polyme
  • the fluorescent oligonucleotides are equivalent in yield and length to the non-fluorescent oligomer made with the commonly occurring nucleotides and hybridize to target template DNA and display the same thermal stability and capacity to stain with ethidium bromide as do the nonfluorescent controls once the hybrid duplex has formed.
  • the production of fluorescent oligonucleotides can be taken directly as evidence of the presence of a particular sequence, or the identity can be further established by (i) hybridization with a defined complementary probe, and (ii) sequencing to establish the analogous sequence; and
  • RNA-dependent RNA polymerase will synthesize an RNA molecule using ribonucleotides, e.g., FTP as a substitute for ATP and UTP instead of TTP, which is the analogous complement to one, and only one, of the two strands of the template.
  • ribonucleotides e.g., FTP as a substitute for ATP and UTP instead of TTP, which is the analogous complement to one, and only one, of the two strands of the template.
  • the resulting single stranded probes can be used directly in a subsequent hybridization reaction without a denaturing step.
  • Solid support-bound oligonucleotide which has already been acid washed to deprotect the 5'-OH group, is reacted with 5'-trityl protected deoxynucleoside analog-3'-0- phosphoramidite in anhydrous acetonitrile in the presence of tetrazole under argon, washing away excess reagents, and then oxidizing the phosphite product to the desired phosphate with a solution deprotect the new 5' terminus, the cycle can be repeated as many times as necessary to achieve the desired length and sequence; additional nucleotides which are added may be the commonly occurring nucleotides or they may be additional fluorescent nucleoside analogs.
  • one or more fluorophores may be incorporated within a given probe up to and including complete substitution of, e.g., all of the A residues in a desired sequence with formycin residues.
  • the couplings can be performed manually in a minireactor vial, utilizing a 10 minute coupling time, or on a Pharmacia LKB Gene Assembler or similar instrument utilizing the programmed synthesis protocols.
  • the fluorescent oligonucleotide is then isolated by cleaving the DNA from the porous glass support by incubation at 55°C overnight in NH 4 OH:ethanol (3:1).
  • the fluorescent DNA containing ammonium hydroxide solution can then be quickly dried in a Speed- Vac and then separated from failure sequences of a QEAE-HPLC column using a shallow salt and pH gradient. Yields for the nucleoside analog phosphoramidites are comparable to those obtained with standard amidites based on repetitive yield calculated from trityl cation release at the deprotection step.
  • Figure 16 depicts the invention scheme used to make the 2'-deoxy-5'-triphosphate or 2'- deoxy-3'-0-phosphoramidite of formycin A While the first phase has been previously accomplished by the reaction with ⁇ -acetoxyisobutyryl halides as described by De Clerq et al ([1987] J. Med Chem. 30:481), the procedure produces both the 3' and 2' deoxy forms which are difficult to separate and are produced in low yield.
  • the present invention employs a 3',5'-disila protection which has previously been applied successfully in the conversion of adenosine to 2'- deoxyadenosine ([1981] J. Am. Chem. Soc. 103:932). The method appears to be generally applicable to the corresponding conversion of many fluorescent nucleoside analogs.
  • 2-deoxyformycin A was treated to attain 5'-0- protection with DMT and benzoylation of the 7-amino group by standard procedures.
  • a solution containing 0.33 mMol of G-cyanoethyl-N,N,N',N'- tetraisopropylphosphorodiamidite was added to 0.3 mMol of the product and 25 mg of diisopropylammonium tetrazolide in 1.5 mL of CH 2 C1 2 was added a solution containing 0.33 mMol of G-cyanoethyl-N,N,N',N'- tetraisopropylphosphorodiamidite. The mixture was mixed for 4 hours and partitioned between CH 2 C1 2 and chilled in saturated NaHC0 3 solution. The CH 2 C1 2 layer was washed with saturated NaCl solution, dried (Na 2 S0 4 ), filtered, and concentrated.
  • RNA oligonucleotides were synthesized from three DNA templates ( Figure 12) using (i) FTP (F 10 s) ⁇ a substitute for ATP, and (ii) a purified E. coli RNA polymerase as originally described by Ward et al ([1969] /. Biol Chem. 12:3242), except that synthesis was allowed to run for three hours at 37°C before the reaction was stopped; FTP effectively replaced ATP but not any of the other three normal nucleotides CTP, UTP, or GTP.
  • reaction products were separated from unreacted reagents by separation at 4°C on Sephadex G-50 in normal saline at pH 7.
  • the scheme for separation of reaction products from unreacted agents is shown as a flow chart in Figure 19.
  • FTP is an effective substrate for RNA polymerase with both native and denatured DNA as well as with synthetic deoxynucleotide polymer templates.
  • samples containing CTP, UTP, GTP, RNA polymerase, one of the DNA templates, and either FTP or ATP a high molecular weight product eluted from either sample in the void volume while the amount of monomeric NTP in the retained fraction from either sample was correspondingly reduced by >70%.
  • 2-diamino-adenosine-5 '-triphosphate were substituted for ATP in the reaction mix, or (ii) the C- nucleosides formycin B-5'-triphosphate (F b TP) or -amino-fo ⁇ nycin B-5'-triphosphate (aF b TP) template.
  • F b TP C- nucleosides formycin B-5'-triphosphate
  • aF b TP -amino-fo ⁇ nycin B-5'-triphosphate
  • RNA dependent, RNA polymerase transcription systems for the synthesis of RNAs for use as substrates and hybridization probes are a fairly common tool of molecular biology. They are uniquely applied here to the development of autofluorescent probes and their production. The method developed is general and applies to any of the phage polymerase systems, including SP6, T7, and T3.
  • the invention employs a pair of promoters which are separately positioned on alternate strands of a duplex plasmid and at opposite ends of a polylinker as shown in Figure 13.
  • the vectors are used to (i) attach promoters capable of effecting asymmetric synthesis through use of a viral polymerase which recognizes one of the promoters, and (ii) replicate multiple copies of a template for use in asymmetric production of a fluorescent probe or of a nonfluorescent copy of the probe target.
  • a copy of the DNA target sequence is inserted into the polylinker in its duplex form and at a restriction site adjacent to one of the promoters.
  • Replication of the plasmid in competent cells provides large amounts of the template for transcription.
  • Two separate but parallel methods have been developed for the asymmetric synthesis of DNA probes. In the first case, ssDNA probes are synthesized from templates which have primer binding site attached at the 5' end of one template strand as shown in Figure 14.
  • the primer may be non-fluorescent or may be synthesized using fluorescent analog phosphoramidites as shown at the right of the Figure.
  • a variation on this is asymmetric amplification and separation in which both strands of a template may be replicated by amplification as fluorescent oligomers, but using a pair of primers in which one, and only one, bears a transient affinity linker such as biotin which may subsequently be used to separate the denatured sense and antisense strands.
  • RNA and DNA probes For both RNA and DNA probes, it has proven practical to establish a reference template, probe sequence, and target sequence against which all transcriptions and probe detection sensitivities are calibrated.
  • the alpha chain oiXenopus translation elongation factor (Xef-l ⁇ ) serves that purpose and asymmetric RNA probe synthesis is used here as representative of all RNA and DNA synthesis.
  • the Xef-l ⁇ mRNA is a major transcription product of the Xenopus embryo which comprises a large percentage of the non-mitochondrial mRNA transcripts that appear immediately after the midblastula transition. The gene for the Xef-l ⁇ was isolated and
  • Example 1 The effective utilization of FTP in the poly d(AT) directed synthesis in Example 1 produced a polymer approximately 300-500 bases in length which, when hydrolyzed and/or sequenced, proved to be a perfectly alternating replicate of the DNA template, but with the sequence: poly (FU).
  • the product could be annealed to like chains by a single thermal cycle, thereby creating the putative product poly (FU):poly (FU); unlike the comparably treated poly (FC), which showed no evidence of self-hybridization as expected, the annealed hybrids of poly (FU):poly (FU) stained with ethidium bromide in agarose gels and gave a sharp thermal transition in both absorbance and fluorescence, proving that the probes could hybridize both effectively and specifically.
  • Example 4 Hybridization of Fluorescent Probes to Target RNAs and Target DNAs: Uses of Linkers to Allow Solid Phase Detection
  • the synthetic template poly (TG) was used to produce the complementary RNA probes poly (AC) and poly (FC), neither of which is self complementary and in which hybrids could not be annealed or detected; of the two only the poly (FC) was fluorescent.
  • a poly (AC) template was amplified using the biotinylated synthetic 22-mer primers, 5# BIOTIN- (TG) n 3' , together with standard polymerase chain reaction (PCR) methods to produce the DNA amplimers having the sequence, 5# BIOTIN-pol)(TG) 3 ', then separated from the unreacted primers by gel sizing and/or QEAE ion exchange chromatography, after which the polymers were radioactively labeled using 32 P-ATP and the enzyme polynucleotide kinase.
  • PCR polymerase chain reaction
  • both of the RNA probes, poly (AC) and poly (FC) formed hybrids which could be characterized by (i) ethidium bromide staining, and (ii) melting behavior; as expected, the fluorescence of the poly (FC) probe was quenched by hybridization.
  • the hybrids could then be adsorbed via the 5 BIOTIN moiety to avidinylated beads, washed to remove unhybridized poly (FC), and equal aliquots assayed for radioactivity and fluorescence.
  • SUBSTITUTE SHEET (RULE 28) amplimers of poly (TG), from Examples 2 and 3, above, as assessed by DNA melting behavior, ethidium bromide staining, and the reappearance if quenched fluorescence following denaturation of the hybrid.
  • Example 6 Assay for Chlamydia trachomatis Using an FTP Substituted RNA Probe Chlamydia trachomatis is an obligatory intracellular pathogen which, in its active infectious stages, contains from 3x10 s to 4x10 s copies of ribosomal RNA (rRNA) and one copy of genomic DNA/bacterium.
  • rRNA ribosomal RNA
  • a primer pair one of which contained a 5'-biotinylated T7 promoter which was 5' to the hybridizing primer sequence, was used to amplify a 150 base pair DNA segment of the MOMP gene from a stock strain of C. trachomatis L2.
  • RNA polymerase promoter Approximately 500 ng of the DNA fragment, which contained the T7 RNA polymerase promoter at the 5' end, was transcribed with T7 RNA polymerase in the presence of rCTP, rUTP, rGTP, and with either rFTP or rATP (+ control). The reaction was stopped by heat inactivating the enzyme for 3 minutes at 100°C. Unincorporated rNTPs were separated from the labeled RNA by gel sizing chromatography on a Sephadex G-25 column, after which the probe concentration was estimated from its absorbance at 260 nm.
  • RNA probe Using a simple dual monochromator fluorescence spectrophotometer, as little as 5 x 10 — 14 moles of the RNA probe could be detected over background when 20 nm slits were used for both excitation and emission monochromators.
  • a photon counting fluorimeter designed for sensitivity is capable of detecting between 5 x 1CT " 16 and 5 x 1(T ⁇ 17 moles of the same probe, equivalent to the amount of ribosomal RNA expected from between 5000 to 50,000 of the bacteria. Two hundred microliters of either (i) C.
  • trachomatis genomic DNA or (ii) the amplified target DNA were mixed with 200 ⁇ L of a 1/200 dilution of the probe in hybridization buffer (0.15 M NaCl, 0.02 M sodium citrate, 0.02 M HEPES, 0.004 M EDTA, pH 7.4) and the mixture boiled for 3 minutes, after which they were allowed to cool slowly to room temperature over one hour.
  • hybridization buffer 0.15 M NaCl, 0.02 M sodium citrate, 0.02 M HEPES, 0.004 M EDTA, pH 7.4
  • fluorescence of the probe may be detected at dilutions of the sample which contain less than 1 x 1CT "16 moles of target DNA which is roughly equivalent to the sensitivity required to detect less than 10,000 bacteria if a single similarly sized probe were used to detect rRNA from infectious Chlamydia.
  • the probe used here is about 150 bases in length, contains approximately 38 formycin residues per probe, and binds only to a single target site on each copy of the ribosomal RNA It is an important feature of this invention that increasing the number of fluorophores in a probe, or probe "cocktail,” also increases the sensitivity of detection.
  • Example 7 - Detection of Multiple Target Sites
  • An important aspect of the asymmetric syntheses to both diagnostic and therapeutic, e.g., antisense, applications of nucleic acid probes is the capacity for concurrent synthesis of probe "cocktails" which may comprise probes which differ in length or differ in the locations or numbers of the target sites on RNA or genomic DNA to which they will bind. Utilization of probe cocktails to three different types of diagnostic targets illustrate the broad importance of this feature.
  • a Single target nucleic acids present in multiple copies. In some species of pathogen, multiple copies of rRNA are present in each organism, e.g., each bacterium of Chlamydia trachomatis contains approximately 2 x 10 4 rRNA molecules per organism.
  • rRNA of Chlamydia is typically between 3000 and 5000 nucleotides in length
  • sensitivity in a diagnostic assay may be increased significantly by use of a probe cocktail specific for target sequences on rRNA and made of as many as 5 to 10 different probe sequences, each of which can bind to discrete segments of the target rRNA or target DNA as indicated with probes (a) to (e) in the lower half of the diagram shown in Figure 20 in which (a), (b), (c), (d), and
  • (e) are analogous complementary probes specific for different target sequences of a single DNA strand.
  • rRNA sequences are highly conserved, hence only short variable sequences are useful for the detection and identification of infectious pathogens.
  • diagnostic sensitivity is that only limited numbers of
  • 'reporter' labels can be used on each probe, thereby limiting sensitivity; and (ii) only a few pathogens carry rRNA in high copy numbers, and many, such as the DNA viruses, carry no rRNA at all, hence the number of diagnostics which can employ this strategy is limited.
  • the genomes of all organisms are significantly larger than rRNA and typically carry more numerous and larger unique segments which can serve as target sequences for nucleic acid probe hybridization.
  • the complete genome of Chlamydia trachomatis has been isolated and consists of a relative small double stranded DNA with a molecular weight of >660 x 10 6 or slightly more than 1 x 10 6 base pairs.
  • Each bacterium also contains a 4.4 x 10 6 dalton plasmid containing >7 kbases.
  • the plasmid is unique to Chlamydia in its entirety— no cross-hybridization can be detected with the DNA from, e.g., Neisseria gonorrhea— -indeed, no cross-hybridization occurs between the different restriction fragments of the plasmid itself. Even when no other portion of the Chlamydia genomic DNA is chosen for use as hybridization targets, a cocktail specific for the multiple restriction fragments of the Chlamydia plasmid alone is equivalent in length to more than 4 Xef-l ⁇ probes and can be detected at levels equivalent to between 100 and 1000 bacteria.
  • Ribosomal gene repeats are of particular interest in the kinds of DNA based diagnosis described in this invention. Like the ribosomal genes, they are present in high copy numbers, which improves sensitivity of detection but, in addition, the spacer regions between genes are normally highly variable from species to species, since they are not subject to selective pressures.
  • Multiple copies of the same unique sequence on a single DNA strand represents a special case in which the hybridization targets are a cocktail of loci on each genome; that is, a single probe sequence can probe multiple target sites of the same sequence and on the same DNA strand. They are ideally suited as species and genus specific probe targets.
  • Genomic DNA from E. tenella was digested with several different restriction enzymes, and the fragments ligated into appropriately cut asymmetric plasmid vectors and were used to transform Escherichia coli. Colonies were screened for repeat sequences by hybridization with Eimeria tenella genomic DNA that had been labeled with 35 S by random priming. Strongly hybridizing clones were picked and subjected to differential screening with labeled genomic DNA from E. mitis, E. maxima, E. acervulina, and E.
  • the entire sequence of the insert in the latter clone contains 334 base pairs. Physical characterization of the restriction fragments indicates that the sequence is present in tandemly repeated units of approximately 738 base pairs and that a minimum of 30 genes are tandemly linked and all appear to be on one chromosome. Asymmetric probes synthesized using the tandem repeat as a template contain 179 formycin A residues per template sequence.
  • tandem repeat targets extends well beyond sensitivity, however, or simply the detection of this single genus, since tandem repeat sequences appear in a genomic DNA of a wide variety of species and genera, and are distinct for those species, thereby providing a broad basis for the design of diagnostic assays for a wide variety of pathogens, including those for which no rRNA targets exist.
  • Example 8 The Use of Non-Specific and Non-Hybridizing Fluorescent Oligomers as Universal Fluorescent "Tags" by Ligation or Chemical Linkage
  • Simple modification of the template to produce a "sticky end" at the 3', 5', or both 3' and 5' termini e.g., to 5' ACGT-polyd(AT), polyd(AT)-TGCA 3' , or 5 ⁇ CGT-polyd(AT)-TGCA 3' , respectively, enabled synthesis of nucleic acid probes with all of the above properties, but which could also be ligated, either (i) to like strands to produce longer fluorescent probes, or (ii) to other hybridization sequences universal label for any cloned DNA fragment, and allows a given probe to be identified by two non- hybridizing but highly fluorescent sequences at its termini, without the need to denature the hybrid for detection as was seen with the simple poly (FU) probe, above.
  • FU simple poly
  • Equivalent non-hybridizing universal probes can be readily made by chemical synthesis using, e.g., the etheno analog phosphoramidites, e.g., l,N 6 -ethenoAdenosine-3 '-O-phosphoramidite (eA), to synthesize non-specific tags which can subsequently be linked to any hybridization probe.
  • the etheno analog phosphoramidites e.g., l,N 6 -ethenoAdenosine-3 '-O-phosphoramidite (eA)
  • the 3' or 5' termini of such universal probes can also be prepared for chemical, rather than enzymatic attachment to other oligomers or solid phases, through the addition of, e.g., 5'-amino hexyl, 5'-sulfhydryl hexyl, 3'-aminohexyl amino, N-hydroxysuccinimide esters, and other such linkers.
  • 5'-amino hexyl 5'-sulfhydryl hexyl
  • 3'-aminohexyl amino, N-hydroxysuccinimide esters, and other such linkers e.g., 5'-amino hexyl, 5'-sulfhydryl hexyl, 3'-aminohexyl amino, N-hydroxysuccinimide esters, and other such linkers.
  • sustained signal amplification is non-quantitative and can be useful for a situation where extreme sensitivity is required to answer "yes” or “no” whether a particular gene marker is at all present, for example, where low copy numbers of a target sequence are present.
  • sustained Signal Amplification is described in more detail in Example 8(B), below.
  • Homopolymers of non-hydrogen bonding fluorescent nucleoside analogs can be used together with asymmetric synthesis of ssDNA and RNA to increase the density of fluorescent labeling on cocktails of small probes, on small fragments as in sequencing, and to increase sensitivity of labeling of small or low copy number target.
  • the general concept comprises an oligomeric probe constructed along a typical phosphodiester backbone, but which can be divided into distinct functional regions-the 5' fluorescent homopolymer; primer, or promoter complement; an optional "tether" region, which can connect the homopolymer to the primer, and a target complement.
  • a diagram of this described 5' universal end label is shown in Figure 22.
  • the functional regions of the phosphodiester chain as shown in Figure 22 are:
  • A a non-base pairing homopolymer of from 1 to about 50 fluorescent nucleotide analogs
  • B an optional non-nucleotide phosphodiester "tether" comprising, e.g., one or more freely rotating alkyl chains inserted as part of the phosphodiester backbone of the oligomer;
  • C an enzymatic synthesis primer for use in initiating enzymatic synthesis of the target-specific D region.
  • Representative examples would be the complementary sequences to the T7 RNA polymerase promoter or to the M13 forwared primer as are used in asymmetric RNA or DNA probe systhesis described herein;
  • Regions A, B, and C typically from 20 to 60 bases in length, can be chemically synthesized.
  • the 5' universal end label comprises at least regions A and C and, undley, can also include the optional region B.
  • D a target complementary sequence of from 40 to 20,000 nucleotides in length. This sequence may or may not include fluorescent nucleotide analogs, but functions primarily as the region which synthesized from templates adjacent to the promoter or primer site to which region C is complementary. The entire 5' universal end label can be used as the primer for DNA or RNA replication of the target- specific complement.
  • Enzymatic synthesis using a 5' universal end label is illustrated using the M. tuberculosis IS6110 template (a sequence unique to the bacterium) which has been inserted into a standard Gemini plasmid to create a synthesis template. Other plasmids can be used as well. This enzymatic synthesis process is shown in Figure 23. The following advantageous properties of the 5' universal end label have also been discovered.
  • the excitation spectrum of one non-hydrogen bonding fluorescent analog, ethenoadesosine (designated F 185 ), is compared with the comparable excitation spectrum of formycin (F 105 ).
  • F 185 extends further into the UV wavelengths.
  • Two important discoveries have been made about both the excitation and emission spectra of F 185 : (i) the wavelength maxima are the same at both pH 7 and pH 11, and (ii) the quantum yield is more than lOx that of F 105 having values of 0.55 and 0.65 at pH 7 and 11, respectively. This allows the use of the 5' universal end label under a wider variety of pH conditions and can result in significantly greater luminescence from fewer total fluorophores.
  • an F lg5 20-mer which is excited at pH 11 over the range 270nm ⁇ A ⁇ 310nm can be equivalent to labeling with between 3 and 10 fluorescein molecules. Furthermore, the fluorescence does not quench and can be used with time resolved spectroscopy.
  • non-base pairing end labels do not interfere with primer-mediated DNA amplification or replication, are water soluble at concentrations up to 10 "3 M, and do not increase the background in a binding assay due to non-specific hybridization to non-target sequences.
  • the 5' universal end label can be used to increase sensitivity of detection by using a cocktail of relatively short probes for which the length of the "D" region is approximately 100 bases.
  • the 1361 bp IS6110 sequence of M. tuberculosis has been used as a target for a cocktail of 10 probes, each having a different "D" segment or complementary target sequence.
  • Each probe bears the same 5' universal end label.
  • each bacterium has the potential for being labeled by a probe cocktail with permanent fluorophores which are equivalent in instantaneous emission to between 480 and 1600 fluorescein molecules.
  • Hepatitis B presents such a case.
  • the entire genome of Hepatitis B virus (HBV) is only 3200 bases long and, in the virion, one of the strands is even shorter.
  • the virion contains a DNA polymerase which utilizes nucleotide triphosphates from a host cell to complete the short chain as the first step in an infection.
  • the DNA polymerase of the virion utilized together with a novel fluorescent nucleoside analog described herein was combined with a non-PCR type of amplification which has heretofore been used only for RNA replication.
  • the virion DNA serves as an in situ template and, in combination with the above-described asymmetric synthesis method, can be used to amplify the intensity of the fluorescent signal.
  • the process which may be better understood by referring to Figure 27, involves two steps.
  • the sample DNA is combined with (i) deoxynucleotide triphosphates (deoxyNTPs) including triphosphorylated fluorescent nucleoside analogs, and, (ii) two primers (shown as A and B in Figure 27), the first of which has at its 5' end a sequence complementary to an RNA polymerase promoter.
  • deoxyNTPs deoxynucleotide triphosphates
  • two primers shown as A and B in Figure 27
  • the primers referred to in this Example are described as "A” and “B” to indicate the use of two separate primer. These are described as such for illustrative purposes and, as such, would be understood by those in the art to refer to any primer which comprises a sequence complementary to a promoter region on the target sequence and which can be used with a nucleic acid polymerase.
  • the T7 RNA polymerase promoter is designated by the thicker line at the end of primer A
  • the sample is first incubated at 37°C for 10 minutes to allow the viral DNA polymerase to complete the short genomic strand; the sample is then raised to 65°C for 1 minute to denature the genome, after which the primers are annealed at 42°C.
  • the two enzymes, reverse transcriptase and T7 RNA polymerase are added, together with the riboseNTPs including the fluorescent ribonucleoside analogs, and the entire sample is incubated at 42°C for 1 hour. This creates a cycling synthesis of DNA strands and RNA strands as indicated in the lower half of Figure 27.
  • the net effect is to produce somewhere between 10 8 and 10 9 fluorescent RNA strands and about 100-fold less fluorescent DNA strands.
  • the sample can be simply read for fluorescence to determine whether any template, in this case Hepatitis B DNA, was present in the sample.
  • a novel method for detecting fluorescent nucleoside analogs, fluorescent oligonucleotides or analogous sequences, of the amount of bound fluorescent oligonucleotide probe has been developed based on the use of photon counting to measure the amount of a fluorophore in a sample and is described herein below.
  • the method differs from time resolved spectroscopy in that the method integrates all fluorescence emission from a fluorophore or nucleic acid probe, independent of the wavelength of the emission and is both a novel combination of time and spectral integration and a novel application of photon counting to the identification, detection, and quantitation of nucleic acid target sequences to diagnostic assays and therapeutic treatments.
  • the fundamental experimental parameter used in any measurement of luminescence is the intensity of the luminescence, /, the units of which are moles of photons per second per liter. Because the fluorescent nucleoside analogs used here are, for all practical purposes, permanently fluorescent and do not photobleach within the lifetime of a typical measurement, the luminescence of fluorescence, measured in moles of photons emitted per second per mole of fluorophore, can be used as an index of the amount of fluorophore, and hence probe, in a sample.
  • the preferred instrumentation for such measurements comprises (i) a 150 watt Hg Xe CW cylindrical lamp capable of high intensity excitation over the range 290 nm ⁇ ⁇ ⁇ 320 nm, (ii) an ultrahigh sensitivity photomultiplier in which the photodynode is coated to allow a response only over the range of emission 360 nm ⁇ A ⁇ 550 nm, (iii) a cylindrical cuvette with quartz excitation windows but glass walls which can serve as the emission filter.
  • the cuvette is mounted so that the entire sample can be collected at the face of the photomultiplier tube, and (iv) 5 computer-driven photon counting clocks, connected in seriatim, and each capable of discriminating between photons at a frequency of 10 9 per second.
  • the chemistries and procedures of the invention can be used to create and characterize any probe synthesized using fluorescent nucleoside analogs, whether the synthesis is enzymatic or chemical, for both fluorescence and hybridization specificity.
  • probes can be used not only in the solution hybridization formats described here, but also in the more frequently used laboratory procedures such as "dot-blot" detection, electrophoresis in agarose or polyacrylamide gels, Southern blotting, and hybridization on filters
  • linkers are not essential to the solution hybridization, any appropriate affinity linker such as biotin/avidin or homo- or heterobifunctional linker can be used to capture the probe or hybrid for purposes of concentration, isolation, or detection, as illustrated for the PCR amplified DNA fragments of Figure 18.
  • the present invention includes linker derivatized fluorescent nucleotides, as well as oligonucleotides, linker derivatized primers for use in amplification and subsequent detection with fluorescent oligonucleotide probes, oligonucleotide probes, plasmids, and therapeutics made or otherwise "tagged” therefrom, and/or their uses and applications such as are described herein.
  • Such derivatizations include, but are not limited to, transaminations to purine or pyrimidine nucleosides and/or their fluorescent structural analogs, amino-thiol, azido-, aldehyde, hydroxysuccinimide, 5' aminoalkyl-3'-0- phosphoramidite, 5'-thioalkyl-3'-0-phosphoramidite, 3'-aminohexyl amino, amino silanes, and aminosilyl derivatives and other such linkers and groups reactive with linkers or in condensation reactions such as Schiff base condensations of 3' or 5' oxidized ds-diols, as are familiar to one skilled in the art. To illustrate this a specific case is offered:
  • a set of non-fluorescent amplification primers for the MOMP gene sequence was chemically synthesized; at the end of synthesis an additional cycle was used to add 5'- aminohexyl-3'-0-phosphoramidite to the 5' terminus of the completed primer with the addition chemically synthesized, using standard phosphotriester chemistry, (ii) Following cleavage from the solid phase support in strong ethanolic base, the terminal amino group of each strand was reacted with NHS-biotin ester to provide the 5' biotinylated primers.
  • the captured amplimers were hybridized with fluorescent analog labeled oligonucleotide probes as described above and the amount of target sequence in the amplimers quantified. Included in the present invention are such attachments of fluorescent oligonucleotides to other fluorescent or non-fluorescent oligonucleotides to immobilizing beads, filters, or activated plastic plates and done through enzymatic attachment such as ligation, or chemical attachment through such linkers as are described herein.
  • Oligonucleotides can be synthesized or derivatized as described herein which have two or more spectrally distinct, detectable labels, either by using two or more nucleoside analogs with discrete fluorescence emission characteristics, or by use of a covalently attached FRET acceptor, such as is described hereinabove. FRET acceptors can also be used to enhance or broaden the sensitivity of the probe emission.
  • the excitation spectra of such dyes as the coumarins e.g., 7-amino-4- methylcoumarin-3-acetate, 7-methyl-umbelliferone, the naphthalene and anthracene dyes, etc.
  • Such dyes as 7-amino-4-methylcoumarin-3-acetate may thus be used either (i) as a covalently attached FRET acceptor, e.g., by reacting the N-hydroxysuccinimide ester with prescribed amino groups on the oligomer, or (ii) by simply adding the dye to a solution of the probe to act as a FRET indicator of probe fluorescence.
  • this methodology allows amplification of the probe signal through more efficient capture of the emitted light, reduction of background light due to light scattering from excitation sources, and detection at longer visible wavelengths.
  • RNA fluorescent probe is contacted with a DNA sample.
  • the RNA fluorescent probe hybridizes to a target DNA sequence.
  • RNase H only digests RNADNA hybrids, not ssRNA probes.
  • the resulting fluorescent monomers are released into solution, and a second RNA probe can hybridize to be digested.
  • the monomers are separated from probes on standard membranes, and the amount of monomer released is measured by simple fluorometiy. The specimens with no DNA for hybrids will show no fluorescence.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Chlamydia trachomatis
  • C INDIVIDUAL ISOLATE: L2/434/Bu
  • G CELL TYPE: Bacterium
  • MOLECULE TYPE transcribed DNA or RNA
  • MOLECULE TYPE transcribed DNA or RNA

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Abstract

On a identifié des analogues structurels des six N-nucléosides non fluorescents que l'on trouve habituellement dans l'ARN et dans l'ADN, fluorescents de façon inhérente dans des conditions physiologiques et on a mis au point des procédés servant à les préparer. Ces analogues peuvent être incorporés dans des oligonucléotides d'ADN et/ou d'ARN par l'intermédiaire d'une synthèse enzymatique ou chimique, de manière à produire des oligonucléotides fluorescents possédant des séquences prescrites. Ces séquences analogues peuvent être identiques à des séquences d'ADN ou d'ARN gabarit ou cible, ou représenter le complément analogue desdites séquences, auxquelles peuvent s'hybrider les oligonucléotides fluorescents. L'invention concerne également des procédés de préparation de sondes d'oligonucléotides d'ARN ou d'ADN de l'invention, des intermédiaires utilisés dans lesdits procédés et des procédés d'utilisation des sondes décrites dans des méthodes d'amplification, de détection, d'identification et/ou d'hybridation d'oligonucléotides.
PCT/US1994/009316 1993-08-18 1994-08-18 Applications de n-nucleosides fluorescents et d'analogues structurels fluorescent de n-nucleosides WO1995005391A1 (fr)

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WO1996028460A1 (fr) * 1995-03-14 1996-09-19 Boehringer Mannheim Gmbh Derives de c-nucleosides et leur utilisation dans la detection d'acides nucleiques
WO1997012896A1 (fr) * 1995-10-04 1997-04-10 Epoch Pharmaceuticals, Inc. Oligonucleotides complementaires a liaison selective
WO1997043298A1 (fr) * 1996-05-15 1997-11-20 Research Corporation Technologies, Inc. Nouveaux analogues de nucleosides a groupes aromatiques polycycliques fixes, procedes de synthese de ceux-ci et leurs utilisations
US5808035A (en) * 1995-12-08 1998-09-15 Usher; David A. Protected nucleoside and method for its synthesis
US6218108B1 (en) * 1997-05-16 2001-04-17 Research Corporation Technologies, Inc. Nucleoside analogs with polycyclic aromatic groups attached, methods of synthesis and uses therefor
EP2045337A1 (fr) 1998-11-09 2009-04-08 Eiken Kagaku Kabushiki Kaisha Procédé pour la synthèse d'acides nucléiques
EP2253717A1 (fr) 2000-04-07 2010-11-24 Eiken Kagaku Kabushiki Kaisha Méthode d'amplification d'acides nucléiques en utilisant comme matrice des acides nucléiques double-brin
US8080381B2 (en) 2003-04-02 2011-12-20 Canon Kabushiki Kaisha Infectious etiologic agent detection probe and probe set, carrier, and genetic screening method
US8140148B2 (en) 1998-01-20 2012-03-20 Boston Scientific Scimed Ltd. Readable probe array for in vivo use
US8431336B2 (en) 2000-07-07 2013-04-30 Diagnostics For The Real World, Ltd. Binding interactions in dipstick assays
EP3808843A1 (fr) 2017-09-14 2021-04-21 Zhongke Xinray (Suzhou) Biological Science Technologies Co., Ltd. Procédé et kit de synthèse d'acide nucléique dans des conditions de température constantes
WO2022226870A1 (fr) 2021-04-29 2022-11-03 中国科学院大学宁波生命与健康产业研究院 Procédé de synthèse d'acide nucléique dans des conditions de température constante, kit et application

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EP0235301A1 (fr) * 1985-09-09 1987-09-09 Teijin Limited Derives de nucleotides de pyridopyrimidine
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996028460A1 (fr) * 1995-03-14 1996-09-19 Boehringer Mannheim Gmbh Derives de c-nucleosides et leur utilisation dans la detection d'acides nucleiques
WO1997012896A1 (fr) * 1995-10-04 1997-04-10 Epoch Pharmaceuticals, Inc. Oligonucleotides complementaires a liaison selective
US5912340A (en) * 1995-10-04 1999-06-15 Epoch Pharmaceuticals, Inc. Selective binding complementary oligonucleotides
US5808035A (en) * 1995-12-08 1998-09-15 Usher; David A. Protected nucleoside and method for its synthesis
WO1997043298A1 (fr) * 1996-05-15 1997-11-20 Research Corporation Technologies, Inc. Nouveaux analogues de nucleosides a groupes aromatiques polycycliques fixes, procedes de synthese de ceux-ci et leurs utilisations
US6218108B1 (en) * 1997-05-16 2001-04-17 Research Corporation Technologies, Inc. Nucleoside analogs with polycyclic aromatic groups attached, methods of synthesis and uses therefor
US8140148B2 (en) 1998-01-20 2012-03-20 Boston Scientific Scimed Ltd. Readable probe array for in vivo use
EP2045337A1 (fr) 1998-11-09 2009-04-08 Eiken Kagaku Kabushiki Kaisha Procédé pour la synthèse d'acides nucléiques
EP2287338A1 (fr) 1998-11-09 2011-02-23 Eiken Kagaku Kabushiki Kaisha Procédé pour la synthèse d' acides nucléiques.
US9909168B2 (en) 1998-11-09 2018-03-06 Eiken Kagaku Kabushiki Kaisha Method of synthesizing nucleic acid
EP2253717A1 (fr) 2000-04-07 2010-11-24 Eiken Kagaku Kabushiki Kaisha Méthode d'amplification d'acides nucléiques en utilisant comme matrice des acides nucléiques double-brin
US8431336B2 (en) 2000-07-07 2013-04-30 Diagnostics For The Real World, Ltd. Binding interactions in dipstick assays
US8080381B2 (en) 2003-04-02 2011-12-20 Canon Kabushiki Kaisha Infectious etiologic agent detection probe and probe set, carrier, and genetic screening method
EP3808843A1 (fr) 2017-09-14 2021-04-21 Zhongke Xinray (Suzhou) Biological Science Technologies Co., Ltd. Procédé et kit de synthèse d'acide nucléique dans des conditions de température constantes
WO2022226870A1 (fr) 2021-04-29 2022-11-03 中国科学院大学宁波生命与健康产业研究院 Procédé de synthèse d'acide nucléique dans des conditions de température constante, kit et application

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