WO1999033867A2

WO1999033867A2 - Modified nucleic acid analogues

Info

Publication number: WO1999033867A2
Application number: PCT/EP1998/008317
Authority: WO
Inventors: Frank Bergmann; Herbert Von Der Eltz; Rupert Herrmann; Stefanie KÖHLER; Volker SCHLÜTER
Original assignee: Roche Diagnostics Gmbh
Priority date: 1997-12-23
Filing date: 1998-12-18
Publication date: 1999-07-08
Also published as: DE19757516A1; WO1999033867A3; EP0996631A1

Abstract

Oligomers comprising at least one non-nucleoside unit containing an acid function with a pKa that is lower than 3.0 are particularly suitable as detection probes for nucleic acids as non-specific interactions are reduced in comparison with unmodified oligomers. Even when the concentration of hybridisation buffers is low, it is possible to obtain high signal dynamics and high sensitivity for a low blank signal. Said oligomers are also suitable as primers for lengthening reactions using polymerases and for transfection.

Description

Modified nucleic acid analogs

The invention relates to a method for the detection of a target containing base sequence in a sample using one or more special hybridization probes, a solid support to which this hybridization probe is bound, and the use of negative charges in partially charged hybridization probes to avoid non-specific binding and to increase the selectivity in binding reactions.

Detection of nucleic acids in samples has gained considerable importance in analytics in recent years. The validity of such tests has proven to be particularly useful in healthcare, where the detection of pathogens, such as viruses and bacteria, in and outside the body, as well as physical conditions, such as genetic predispositions to diseases, is required. This is all the more true since it has been possible to specifically amplify base sequences from the nucleic acids to be detected, for example with the polymerase chain reaction (PCR, US-A-4,683,202). In general, the samples in which the presence of the nucleic acid to be detected are suspected are brought into contact with a hybridization probe, the probe being selected so that it has a base sequence which is essentially complementary to a base sequence of the nucleic acid to be detected. The fact that the probe hybridizes with the nucleic acid is taken as an indication of the presence of the nucleic acid.

First, fragmented, radioactively labeled nucleic acids were used as hybridization probes. However, these had the disadvantage that they were difficult to manufacture and were quite impure. The introduction of automated nucleotide syntheses made it possible to produce short nucleic acids (oligonucleotides) in high purity and with a defined, freely selectable base sequence. The introduction of chemically labeled mononucleoside phosphoramidites made it easy to introduce labein for non-hazardous, non-radioactive detection. More recently, hybridization probes have also been found which surprisingly bind very tightly to nucleic acids, although they do not or do not mix the sugar-phosphate backbone that is typical for nucleic acids together with contain other basic units (e.g. WO 92/20703. EP-0 672 677 and EP-0 700 928). On the one hand, these probes are highly affine to natural nucleic acids with a complementary sequence, but on the other hand they are also highly selective. From Angew. Chem. 1996, 108, 17, 2068-2070), PNA are known, in the backbone of which a glycine unit is replaced by a glutamic acid unit. This increases the solubility of the probe. It was also found that replacing glutamic acid with lysine did not significantly change the melting point depression (ΔT _m ) of the fully complementary PNA / DNA hybrid compared to a PNA / DNA hybrid with a mismatch. Disadvantages of the known PNA probes are the strong tendency towards non-specific bindings, high blank value signals and a lack of solubility. DNA probes have the disadvantage that the renaturation of an amplicon duplex competes with the hybridization of the (D) NA probe.

The object of the present invention is to partially or completely avoid the disadvantages of the prior art, in particular to utilize the higher affinity of PNA probes while avoiding non-specific bindings and high blank value signals. Additional suppression of amplicon re-hybridization is also an advantage of the present invention (use of low salt conditions).

The present invention therefore relates to a molecule which can be hybridized with nucleic acids and has a backbone of monomer units linked linearly to one another, at least one of the monomer units being a non-nucleoside unit. which contains an acid function with a pK _a of less than 3.0. The invention also relates to monomers for the production of these molecules. Methods for the detection of nucleic acids using these molecules and the use of acid functions, preferably with a pK_ of less than 3.0, in probes to avoid non-specific binding of the probes to non-complementary base sequences and to solid supports, to increase the selectivity in binding reactions, in primers as well as in molecules for transfection.

In principle, molecules which hybridize with nucleic acids and which contain non-nucleoside units are known. In principle, they can be divided into molecules that contain only (non-natural) non-nucleoside units and molecules that contain both non-nucleoside and (naturally occurring) nucleoside units. - All these molecules have in common that they are composed of monomer units. These are linearly linked with one another in such a way that an oligomer with a continuous backbone results, to which ligands are bound at substantially uniform intervals. In order for the oligomer to be able to hybridize with nucleic acids, some of these ligands (generally more than 6 ligands) must be able to recognize and bind nucleobases of the nucleic acid via hydrogen bonding. Such ligands are, for example, the naturally occurring nucleobases, such as A, G, C, T and U, but also artificial heterocycles. such as 7-deazapurine or pseudopyrimidine. In addition, these molecules can contain other ligands that do not necessarily contribute to binding with the nucleic acid. Such ligands are, for example, groups which can be used to detect or separate the molecules in methods for the detection of the nucleic acid. Such ligands can attach to the nucleobases. but can also be bound to the basic structure instead of the nucleobases or in addition to the nucleobases. Examples of such ligands are described, for example, in WO 92/20703, WO 95/14708 (metal complexes), DE 197 12 530.1 and WO 95/16202 (peptides).

The production of molecules hybridizing with nucleic acid, in which the basic structure is composed of a sequence of monomer units linked via peptide bonds, is described, for example, in WO 92/20702. In a first aspect, the subject matter of the present invention differs from that described therein in that at least one of the monomer units used there contains a function with a pK _a of less than 3.0.

EP-A-0 672 677 describes a synthesis of nucleic acid analogs in which nucleosidic and non-nucleosidic monomer units are mixed with one another. In a further embodiment, the subject matter of the present invention differs from the molecules described therein in that one of the non-nucleoside monomer units contains an acid function with a pK of less than 3.0.

When we speak of a modified monomer unit in the following, we are referring to the non-nucleoside monomer unit, which has the acid function with a pK. less than 3.0 or contains the negative charge. A monomer unit is preferably understood to mean a unit which is part of the above-mentioned oligomer and which contains at most one nucleobase. These units preferably contain only those parts of the molecule which were already present in the monomers used for the synthesis of the oligomers. As a rule, the oligomer is between 6 and 100, preferably 8 and 30, monomer units long. In the following, the calculation of the length of the basic unit of each monomer is based on the fact that the geometric length between the connection points of the individual monomers is essentially the same and preferably with the connection points of the monomers. from which the oligomer is made coincides. For example, in the preferred case of peptide nucleic acids, the length of a backbone unit extends from the carbonyl group of one end of the monomer unit to the amino end of the same monomer unit which is bonded to the subsequent carbonyl group of the next monomer unit. The length of a basic framework unit defined in this way is preferably between 4 and 8. particularly preferably 6 atoms.

The modified monomer unit can in principle be any, ie also the last, nucleobase-containing monomer unit of the molecule. However, the modified monomer unit is particularly preferably not the last base-containing monomer unit of the oligomer. The modified monomer unit is particularly preferably within the inner third of the total length of the molecule.

The molecule hybridizing with nucleic acids can in principle contain as many modified monomer units as there are monomer units. However, it is preferred that less than half of the monomer units represent a modified monomer unit. The remaining monomer units are preferably not negatively charged; they are preferably neutral. The discrimination between nucleic acids with a different base sequence in one or more positions is favored by the fact that the molecule is selected so that the modified monomer unit is installed approximately in the middle of the molecule, specifically to the nucleic acid to be detected, but not with a nucleic acid to be discriminated therefrom is complementary in this position (mismatch).

A nucleoside unit is understood to mean a unit that contains both a sugar portion and a phosphate portion. A non-nucleoside unit is thus a monomer unit in which the base does not adhere to a sugar portion, and / or the Sugar portion is not bound to a neighboring monomer unit via a phosphate residue.

For the purposes of the present invention, an acid function is understood to mean a chemical group which is covalently bound to the non-nucleoside unit. In the form bound to the non-nucleoside unit, this group preferably has a pK of less than 3.0. 2- or polybasic acids are preferred. Preferred acid functions are selected from the group of inorganic acids, in particular the groups of sulfonic acids, phosphonic acids or phosphoric acids or their salts. A sulfonic acid residue is preferably a group of the formula -SO ₃ H. A phosphonic acid residue is preferably understood to mean a residue of the formula -PO ₃ H ₂ . Phosphoric acids contain the group -OP (OH) ₂ O. The acid function is preferably not involved in the covalent bond between two monomer units. In particular, the acid function is preferably bound to a carbon atom of the backbone, which is not the first in addition to a hetero atom (O, P, S or N) used for the linkage with the next monomer. The acid function can either directly or via a spacer A 'with the basic unit of the monomer unit, preferably one atom of the continuous chain of atoms in the backbone, e.g. B. a C atom. be connected. The acid function together with the spacer A 'is also referred to below as a group R containing an acid function with a pK _a of less than 3.0.

The spacer A 'connects an atom of a series of linearly consecutive atoms of the basic structure with an atom of the acid function. In principle, these spacers can be between 1 and 20 atoms long. The spacer A 'particularly preferably has the formula III

- (- (- A'-) _a - (- A ² -) _b - (- A ³ -) _c -) _d - (III),

in the

A ¹ oxygen. NR ⁶ or sulfur,

A ^{2 is} a substituted or unsubstituted alkylene, alkynylene or alkenylene

Rest. A ³ oxygen. Sulfur or NR ⁶ ,

R ^{6 is} independently hydrogen. Acyl or alkyl,

a and c independently of one another a number from 0 to 2,

b is a number from 1 to 10, and

d is a number from 1 to 4

mean, the spacer can be installed in either of the two possible orientations.

Alkyl. Acyl, alkylene. Alkinylene and alkenylene radicals in the definition of A ² and R ⁶ preferably have 1 to 3 carbon atoms. Substituents can be chosen, for example, from the group hydroxyl, halogen (e.g. Cl, Br or F) or amino.

The molecule according to the invention is preferably an oligomer hybridizing with nucleic acids and containing at least one non-nucleoside monomer unit of the formula I.

[1]

(I)

wherein

L is selected from the group hydrogen, hydroxyl, (C, - C ₄ ) - alkanoyl, nucleobase-binding group, (C ₆ -C ₁₄ ) aromatics, heterocycles with nitrogen, oxygen or / and sulfur atoms, intercalators and reporter groups,

A is a spacer with a length of 1 to 3 atoms.

x is a number between 0 and 3,

B is a basic unit with a length of between 4 and 8 atoms, R is a group containing an acid function with a pK_ of less than 3.0

mean. A preferred alkanoyl group (acyl group) is the acetyl group. Nucleobases are to be understood as the naturally occurring bases A, C, G, T and U. A nucleobase-binding group is a group which can form with such a nucleobase via interactions specific to hydrogen bonds. In addition to the (complementary) natural nucleobases, this also includes unnatural nucleobases, such as. B. 7-deazaguanosine or pseudopyrimidines. Aromatics can be both substituted and unsubstituted. Particularly suitable substituents are hydroxyl, C 1 -C ₂ -alkoxy or amino. Preferred aromatics are the phenyl, pyrenyl and naphthyl radicals. Heterocycles are cyclic compounds that contain one or more heteroatoms. selected from the group consisting of O, N and S, which can be non-aromatic or aromatic. They too can be substituted by the radicals mentioned for aromatics. Examples of heterocycles are the pyridyl radical and the piperidyl radical. Intercalators are molecules that can be inserted into the base-base interaction of neighboring bases, e.g. B. acridine. A reporter group can be used to detect or immobilize the molecule. Immunologically active compounds and biospecific interactions from binding molecules from reporter groups are particularly suitable. A particularly preferred group of reporter groups are the markings or labels. The electrochemiluminescent ruthenium bispyridyl complexes are particularly preferred here.

L is particularly preferably bonded via a spacer A to a nitrogen atom, particularly preferably to an amine nitrogen atom, in B. The group R is likewise preferably bound to the backbone unit, but not directly to the nitrogen atom to which L is bound.

Particularly preferred molecules have at least one monomer unit of the formula II

wherein L. A and x have the meanings given above and

E 'and F' independently CO, CS. SO. SO ₂ or NR ¹ ,

where R is selected from the group hydrogen and (C, -C ₃ ) - alkyl,

N is an amine nitrogen atom. and

C and D are independently selected from the group (CR ² R ³ ) _n and CR ² R ³ CR ⁴ R ⁵

in which

R ² , R \ R ⁴ and R ^{5 are} independently selected from the group

Hydrogen, C, -C ₆ - (substituted or unsubstituted) alkyl, hydroxyl, carboxyl and a group R, which contains the acid function, and

n is a number between 1 and 4.

At least one of the radicals R ² -R ³ or R -R ⁵ is a group R.

The invention also relates to a molecule of the formula IV

in the

is selected from the group hydrogen, hydroxyl, (C, -C ₄ ) - alkanoyl, nucleobase-binding group in protected or unprotected form, (C ₆ - C) - aromatics. Heterocycles with nitrogen, oxygen or / and sulfur atoms, intercalators and reporter groups.

a spacer with a length of between 1 and 3 atoms,

a number between 0 and 3. E and F independently COOX, CSOX. SO ₂ X. SO, X or NR ¹ Y,

in which

R ^{1 is} selected from the group consisting of hydrogen and (C, -C ₃ ) -alkyl,

X is selected from the group consisting of hydrogen, the protective groups and the activation groups,

Y is selected from the group hydrogen and protective group,

N is an amine nitrogen atom and

C and D are independently selected from the group (CR ² R ³ ) _π and CR ² R ³ CR ⁴ R ⁵

in which

R ² , R ³ , R ⁴ and R ^{5 are} independently selected from the group

Hydrogen, C, -C ₆ - (substituted or unsubstituted) alkyl, hydroxyl, carboxyl and a group R containing an acid function with a pK _a of less than 3.0 in protected or unprotected form, and

n is a number between 1 and 4.

These molecules can be used for the synthesis of the oligomers according to the invention. Particularly preferred are those molecules in which one of the radicals E and F is a protected amino or carboxylic acid function and the other either a reactive group, such as a carboxylic acid or primary amino group, or a corresponding activated derivative, e.g. B. an active ester. An activation group is, for example, 1-oxybenzotriazole. During oligomer synthesis, the acid function is preferably protected with the aid of a protective group, preferably in the form of an ester which can be cleaved without the oligomer being fragmented. Such protective groups are, for example, benzyl and β-cyanoethyl. The synthesis methods known, for example, from chemical oligonucleotide synthesis or the synthesis of peptide nucleic acids (WO 92/20702) can be used analogously to the synthesis of the oligomers according to the invention or those used according to the invention. A particularly suitable monomer is shown in Figure 1 (compound 3). It is a derivative of the amino acid homoserine. This compound is at the amino end of the backbone monomer unit with a t-butyloxycarbonyl (Boc) protective group on any exocyclic amino groups of the base with a benzyloxycarbonyl (Z) group and at the hydroxyl group of the homoserine by replacing the hydrogen with a di- Benzyl ester protected phosphate group.

FIG. 1 also shows a compound (4) in which the amino group of the basic unit is protected with a monomethoxytrityl protective group and the exocyclic amino functions of the base are protected with an acyl protective group (benzoyl, isobutyryl). The homoserine hydroxyl group is esterified with a di-β-cyanoethyl-protected phosphate residue. Compound 5 shows the corresponding deprotected monomer unit built into the oligomer. FIG. 1 also shows the corresponding monomer 1 based on serine as the amino acid and the deprotected monomer unit 2. As compounds 6. 7, 8 and 9, the phosphonates and sulfonates based on homoserine are shown. The spacers between the acid function (PO ₄ ^{2 "} , PO ₃ ^{2 '} and SO ₃ ^" ) and the carbon atom of the linear backbone chain preferably follow the formula (CH ₂ ) _n , n taking values from 1 to 6, preferably 2 to 4.

The invention also relates to a method for detecting the presence or absence of a nucleic acid in a sample, which is suspected. that it contains one or more non-detectable nucleic acids (nucleic acid (s) to be discriminated), the base sequence of which differs from the base sequence of the nucleic acid in one or more positions, comprising the steps of contacting a probe which contains a base sequence which is to be detected , but is not complementary to the other nucleic acids, and determining whether binding of the probe to a base sequence complementary to the base sequence has taken place, characterized in that the probe is a nucleic acid hybridizable molecule with a backbone of linearly linked monomer units, at least one of the monomer units is a non-nucleoside unit. which contains an acid function with a pK _a of less than 3.0 is used.

The method according to the invention is a special embodiment of the so-called hybridization tests. the basic features of the specialist on the Are known in the field of nucleic acid diagnostics. Insofar as experimental details are not set out below, the entire content is "nucleic acid hybridization". Edited by BD Harnes and SJ Higgins, IRL Press, 1986. B. in Chapters 1 (Hybridization Strategy), 3 (Quantitative Analysis of Solution Hybridization) and 4 (Quantitative Filter Hybridization), Current Protocols in Molecular Biology, Ed. FM Ausubel et al. J. Wiley and Son. 1987, and Molecular Cloning, Ed. J. Sambrook et al. CSH, 1989.

A probe is generally understood to mean a nucleic acid-binding molecule which has been made distinguishable from natural nucleic acids by attaching a chemical group. The chemical group can be an immobilizable group, on the one hand, or a label. This probe contains a base sequence which contains a base sequence which is complementary to a sequence of the nucleic acid to be detected, but not to a sequence of the other nucleic acids which are not to be detected. This sequence is preferably longer than 10 bases. The sequence is preferably continuous and uninterrupted. The length of the sequence is preferably 10 or more bases, particularly preferably 15 or more bases. The probe can also contain further bases which do not influence the discriminatory properties of the probe, since they do not lead to a stronger binding of the probe to a non-detectable nucleic acid than to the one to be detected. In general, this is ensured if these bases are not complementary to a base sequence which is located directly next to a potential binding sequence for the probe on a non-detectable nucleic acid. Such additional bases can be used, for example, to specifically recognize the probe itself via a nucleotide sequence, e.g. B. for use as a universal detection probe.

A label in the sense of the present invention consists of a directly or indirectly detectable group L. Directly detectable groups are, for example, radioactive ( ³² P), colored or fluorescent groups. Metals or metal complexes. Indirectly detectable groups are, for example, immunologically or enzymatically active compounds, such as antibodies, antigens. Haptens or enzymes or enzymatically active partial enzymes. These are detected in a subsequent reaction or reaction sequence. Haptens are particularly preferred. because the oligomers marked with them Detection via a subsequent reaction with a labeled antibody against the hapten can easily be made. The invention is particularly effective for lipophilic labels such as biotin. Fluorescein. Pyrenyl or ruthenium organic complexes.

An immobilizable group is understood to be a chemical residue which can be used to bind the probe to a solid phase. Suitable for this are therefore both activatable residues (e.g. photoactivatable), but also partners of a specific binding pair, such as receptors and associated ligands, partners of an immunological reaction, such as. B. Haptens. Antigens or antibodies, or vitamins or receptors for these vitamins. Haptens or biotin and its derivatives are particularly preferred as the immobilizable group. Biotin is particularly preferred. The solid phase, to which the probe can be bound by means of the immobilizable group, has a corresponding binding partner or a corresponding reactivity. In the case of biotin, the solid phase can therefore have a coating of streptavidin.

An analyte nucleic acid is understood to mean the nucleic acid which is the aim of the detection. These are to be understood as meaning nucleic acids of any origin, for example viroid nucleic acids. viral. bacterial or cellular origin. They can be in solution, suspension, but also fixed to solids or in cell-containing media. Cell smears, fixed cells, tissue sections or fixed organisms are present. The nucleic acids are preferably in solution (in vitro).

The reaction sequence is usually started by making the analyte nucleic acid available with appropriate reagents. Changes in the pH (alkaline), heat, repetition of extreme temperature changes (freezing / thawing), changes in the physiological growth conditions (osmotic pressure), exposure to detergents, chaotropic salts or enzymes (e.g. proteases, lipases), contribute alone or in combination to the release of the nucleic acids.

Denaturation of nucleic acids means separation of nucleic acid double strands into single strands. In principle, a multitude of possibilities are available to the person skilled in the art, e.g. B. Treatment by alkali hydroxides, by heat, or by chemicals. A specific detection is understood to mean a method by which, if desired, certain nucleic acids can also be selectively discriminated in the presence of others. Nucleic acids can be detected. However, it is also possible to detect a group of nucleic acids with a partially identical or similar nucleotide sequence. To detect double-stranded nucleic acids, either of the two complementary strands can be included. Therefore, the specific sequence of the probe is preferred to a length of more than 10, preferably more than 15 bases to more than 90, preferably more than 95 and particularly preferably 100% complementary to a sequence of the same number of continuously successive bases of the nucleic acid to be detected. The present invention has particularly good effects for sequences which are particularly rich in purine, ie contain more than 50% purines. It is particularly preferred for sequences which contain 4 or more purines in a continuous row. z. B. G included. In this case, the monomer unit modified according to the invention is preferably within this homopurine range.

A nucleic acid or nucleic acid sequence that is essentially complementary to a nucleic acid means nucleic acids or sequences that can hybridize with the corresponding nucleic acid, the nucleotide sequence of which in the hybridizing region is either exactly complementary to the other nucleic acid or differs in a few bases from the exactly complementary nucleic acid. The specificity depends on both the degree of complementarity and the hybridization conditions.

Two bases are usually complementary to one another if they can form base pairings with one another, which preferably follow the Watson-Crick rule or the Hoogsteen base pairing. The complementarity condition refers to two or more base sequences with directly adjacent bases with a length of 10 or more bases. Exactly complementary means 100% complementary: if there are differences, these should be less than 10%, preferably less than 5%, of the bases within the hybridizing region. The differences must of course be chosen so that they do not significantly impair the specificity of the test for the nucleic acid to be detected. The nucleic acid to be detected can be the analyte nucleic acid itself. However, it can also be a nucleic acid which was prepared from the analyte nucleic acid by pretreatment in the sample liquid. Known pretreatments include, in particular, fragmentation, for example using restriction enzymes, or cDNA synthesis from RNA. So that the method according to the invention can fully develop its advantages, it has proven expedient if the nucleic acid to be detected has a size of at least 40 bp. The nucleic acid to be detected is preferably the product of an upstream specific or non-specific nucleic acid amplification. Such nucleic acid amplification methods are known for example from EP-A-0 201 184, EP-A-0 237 362, EP-A-0 329 822, EP-A-0 320 308 or WO 88/10315. In these methods, the analyte nucleic acid serves as a template nucleic acid for the production of the nucleic acid that is ultimately to be detected. However, the nucleic acid can also be a nucleic acid produced by cloning and in vivo propagation.

For example, a method for detecting a specific virus, e.g. B. HGV, in a body fluid (z. B. serum) contain as a first step the lysis of the virus envelope. Methods for the lysis of cell walls are known to the person skilled in the art. For example, the lysis can be carried out by treatment with alkali hydroxide solutions. The addition of auxiliary substances, e.g. B. detergents is possible. This can be followed in particular by viruses which are only present in low concentrations in body fluids (eg hepatitis C virus), in vitro nucleic acid amplification (eg via the polymerase chain reaction or the other amplification methods mentioned above ). A large number of nucleic acids are formed with the analyte nucleic acid as template nucleic acid, which are then detected in the method according to the invention.

If bacteria, for example in food, are detected, the method according to the invention could also be preceded by several steps. In general, bacterial samples are disrupted if necessary after in vivo multiplication of the bacteria under conditions which bring about the lysis of the bacterial cell wall (eg proteinases. Alkali). In the case of samples with a very low concentration, an in vitro amplification of the bacterial analyte nucleic acids can follow. The result of the various pretreatments is a sample liquid which contains nucleic acids in solution and which may contain the reagents used in the preparatory steps and possibly destroyed cell components.

Methods known to the person skilled in the art are available for carrying out the specific detection using the probe according to the present invention. These are based essentially on the fact that the nucleic acid-containing sample is incubated with the probe under conditions in which the probe specifically binds to the nucleic acid to be detected, but not or to a lesser extent to the nucleic acids in the sample not to be detected. Although in most cases the reaction conditions for the hybridization which are suitable for the unmodified probes can also be applied to the molecules according to the invention (for PNA, for example, the conditions mentioned in WO 92/20703), it has nevertheless proven to be the case that the sensitivity and dynamics increase with decreasing salt concentration in the hybridization buffer. This is advantageous because at low salt concentrations, DNA-DNA hybridizations become less favorable. This applies in particular to the case of the renaturation of originally double-stranded analyte nucleic acids compared to the hybridization of relatively short oligomers according to the invention. The probes according to the invention show an inverted image compared to DNA probes. With DNA probes, the signal dynamics and sensitivity increase with increasing salt concentration. For the preferred case of PNA probes modified according to the invention, it has been shown that the preferred concentration of salt (NaCl) is between 0 and 150 mM. A comparison of probes according to the invention against DNA probes is shown in FIG. 2. The first diagram shows the influence of sodium chloride concentration on the sensitivity of an HGV test using a ruthenium complex-labeled DNA probe at different salt concentrations. The second diagram shows the same experiment with a PNA probe according to the invention with the same sequence, the ruthenium complex being bound to the PNA backbone via two glutamine residues. and the backbone also contains two glutamine residues. It can be seen that the salt dependency for the DNA probe is different compared to the PNA probe, even though the probe contains negative charges. Especially at low salt concentrations, at which the renaturation of the amplicon double strand is suppressed. the signal dynamics and sensitivity are optimal when using partially negatively charged PNA probes in contrast to DNA probes. The third diagram shows the salt dependency of a probe that contains two lysine residues (positive charges) in the backbone. It can be seen that the signal dynamics and sensitivity are hardly dependent on salt, but the blank value signals are greatly increased, especially at low salt concentrations. In the fourth graph, the salt dependence is an unmodified one. shown in the backbone neutral PNA probe with two glutamic acid residues as a linker to the ruthenium complex. Here too, as with the partially negatively charged PNA probes, signal dynamics and sensitivity increase towards low salt concentrations. the optimal NaCl concentration is 50 mM. However, the blank signal is significantly increased.

After the incubation, it is determined whether or to what extent hybrids have formed from the nucleic acid to be detected and the probe. For the detection, the label on the probe is used, whose presence in the complex of nucleic acid to be detected and the probe is used to indicate the presence of the nucleic acid to be detected. This is preferably done after having separated any excess probe from the hybrid. This can be done, for example, by trapping the hybrid with the help of a solid-phase capture probe. After an incubation period during which the immobilization reaction takes place, the liquid phase is removed from the solid phase (e.g. from the vessel, the porous material or the pelleted beads). The solid phase can then be washed with a suitable buffer.

The amount of the hybrid bound to the solid phase from the nucleic acid, detection probe and capture probe to be detected can be determined in a manner known in principle, e.g. B. in the manner of a sandwich hybridization process. In this method, the hybrid is implemented with the detection probe according to the invention, which is complementary to a different nucleotide sequence of the nucleic acid to be detected than the capture probe, and hybridizes with it. This forms a hybrid of the detection probe, the nucleic acid to be detected and the capture probe on the solid phase. Such a method is described, for example, in EP-A-0 079 139. The detection probe is preferably added to the sample together with the capture probe with the hybridization solution.

For directly detectable groups, for example fluorescence labels. the amount of label is determined fluorometrically. The detectable group is indirectly detectable z. B. a hapten. the hybrid is preferably reacted with a labeled antibody against the hapten, as described analogously in EP-A-0 324 474. The label on the antibody can be, for example, a color or fluorescent label or, preferably, an enzyme label, such as β-galactosidase, alkaline phosphatase or peroxidase. In the case of enzyme labeling, the amount of nucleic acid is monitored via the mostly photometric chemiluminometric or fluorometric monitoring of a reaction of the enzyme with a chromogenic. chemiluminogenic or fluorogenic substrate measured. The measurement signal is a measure of the amount of originally present nucleic acid to be detected and thus possibly of organisms to be detected.

The non-nucleosidic differs particularly preferably. modified basic scaffold unit at a position in which the nucleic acid to be detected differs from one of the non-detectable nucleic acid present in the sample, from at least one of the two adjacent basic scaffold units, which is not intended to mean the base. This can be realized, for example, in that the adjacent basic unit does not have a negative charge or does not have the acid function, or that it is based on another monomer unit. e.g. another amino acid, based in the linear part of the backbone.

In a particularly preferred embodiment, the sample which contains the nucleic acid to be detected in addition to other nucleic acids is treated in such a way that the nucleic acid to be detected is released from cells which may be present and any proteins adhering to it are denatured. This can be followed by isolation of the nucleic acids from this mixture. The nucleic acids freed from any inhibitors of the amplification can then be amplified, e.g. B. be subjected to a PCR. It is preferred to use one of the primers labeled with an immobilizable group. After completion of the amplification, the amplificates are detected by hybridization with a probe according to the invention. The probe is preferably selected such that it hybridizes with the amplificates in a region between the hybridization positions of the primers. The detection takes place after immobilization of the amplificates and hybridization with the probe. For this purpose, magnetic particles can preferably be used which recognize the immobilizable group of the amplificates. The reagent solution with the excess probes is preferably removed before the detection. lσ

The oligomers according to the invention. those with an acid function with a pKa of less than 3.0 are particularly suitable for the detection of mutations, alleles or polymorphisms as well as for the typing or subtyping of microorganisms. The oligomers preferably contain both such an acid function within the binding region of the oligomer with the nucleic acid to be detected and also a negative charge outside the binding region. e.g. at the end (s) or in the linker between the binding area and the marking.

In addition, it has been found that oligomers. in which less than 80. preferably less than 50 and particularly preferably less than 40%, but at least one of the bases is attached to a negatively charged basic unit, e.g. B. one by an acid function with a pK _a of less than 5.0, preferably less than 3.0. modified, bound, bind significantly less non-specifically to nucleic acids (e.g. length markers) that are relatively unrelated in their sequence. You therefore avoid unspecific binding in reactions to bind the probe to complementary base sequences (Table 1). By relatively unrelated nucleic acids are meant nucleic acids which do not contain a continuous and uninterrupted sequence of more than 10, preferably more than 15 bases, which are more than 50% complementary to an equally long uninterrupted and continuous sequence of bases of the probe. Blank values, especially for electrochemiluminescence measurements. are reduced. Compared to unmodified peptide nucleic acids, the oligomers according to the invention are distinguished by an increased selectivity in binding reactions. In contrast to oligomers. where a positive charge was attached to the backbone (e.g. T-Lys-containing PNAs) and neutral PNAs, significantly lower blank value signals were obtained, especially with decreasing salt concentration (Table 1). The oligomers according to the invention are also less prone to non-specific binding to solid phases, e.g. B. hydrophilic surfaces, such as particles or vessels coated with streptavidin. In this case, non-specific binding is understood to mean a binding that is not based on base-base interaction. Neutral and very particularly negatively charged surfaces are particularly suitable for the use of the oligomers according to the invention.

In Angew. Chem. 1996, 108, 17, 2068, PNAs with a glutamic acid-containing backbone are described. Such oligomers can also be used to avoid unspecific binding non-complementary nucleic acids or solid phases can be used. 18mers and 14mers were used as probes in comparative experiments. The probes differ as shown in Table 2.

Table 1 :

Table 2:

In Table 1 it is shown that the partially negatively charged PNA probes are clearly superior to non-charged PNAs in terms of non-specific binding to non-complementary DNA fragments and show a behavior similar to that of a comparable DNA probe with the same base sequence. The left column explains which nucleic acid components were used as the basis for the detection method. In the first case, biotin-labeled HGV primers were used in different concentrations, which do not contain a sequence complementary to the probe. In the second case, biotin-labeled length markers were used, which also show no significant complementarity to the probe. In the third case, HGV amplificates were used in different dilutions. The amplificates contain a region that is 100% complementary to the probe sequence. In the fourth case the amplificate of an HBV test is used. This amplificate has no complementarity to the probe used. In the fifth case, no nucleic acids are added, so that only the non-specific binding of the probe to the surface of the magnetic particle is measured.

The results from Table 1 show that a blank signal is measured even in the absence of nucleic acids in the reaction mixture (case 5). This is similarly small for DNA probes and for PNA which contain negative charges both in the backbone and in the linker. In contrast, the blank signal rises somewhat when the negative charge in the linker is removed. The blank value signal is particularly large in the case of uncharged PNA. This shows that a reduction in the blank value can be achieved with negatively charged PNA probes.

Cases 1 and 2 also show. that unspecific binding to nucleic acids, which are not similar to the sequence complementary to the probe sequence, also contribute to the blank signal. This applies both to the primers that are inevitably contained in the reaction mixture and to controls, such as standards. Here, too, a reduction in the blank value is determined by using negatively charged probes. This contribution was even completely eliminated in the present experiment.

Case 4 shows that in the presence of amplicons. which, for example, can be attributed to the simultaneous amplification of a further analyte in the reaction mixture, a considerable blank value signal is found which can be practically eliminated by using negatively charged probes.

The reaction preferably takes place in a plastic vessel. In particular, it is possible to use vessels which are normally used as sample storage vessels in conventional automatic analyzers. Examples are vessels made of polystyrene, polyethylene or luran. During the treatment with alkali and detergent, the vessel in which the treatment takes place is preferably already in an automatic analyzer, particularly preferably on a sample rotor, which allows a sampling device to access individual sample vessels located on the rotor. Such an automatic analyzer is described for example in EP-A-0 361 830. These oligomers are also suitable. in which less than 80%, preferably less than 50%, but at least one of the bases are bound to negatively charged basic framework units, particularly preferably the oligomers according to the invention. excellent as a primer. provided that they have at one end a group which can be extended by a polymerase, preferably a hydroxyl group. Primer. which are composed of conventional mononucleotide units and one or more nucleoside units at the C end are described in WO 95/08556 and EP-0 672 677. Surprisingly, it has been found that the incorporation of one or more negative charges in the region of the extendable end improves the effect as a primer. Oligomers according to the invention are particularly suitable as primers. which has an extendable hydroxyl group, e.g. B. Nucleosides. and the next monomer unit is a non-nucleoside unit which has an acid function with a pK _a of less than 5.0. preferably contains less than 3.0. These chimeras can be produced with the monomers according to the invention analogously to WO 95/08566.

Amazingly, oligomers can. in which less than 80%, preferably less than 50%, but at least one of the bases are bound to negatively charged basic framework units, particularly preferably the oligomers according to the invention compared to unmodified, neutral PNAs, e.g. B. with so-called transfection reagents, for. B. DOSPER or DOTAP. are better introduced into cells. Such transfection reagents are for nucleic acids from Bioforum 6/96. Page 264. In particular in the form of peptide nucleic acids, which also have the advantage of stability against enzyme digestion and a high affinity of the nucleic acids, the oligomers according to the invention can therefore be used well in antisense technology or as gene probes.

In the case of the oligomers. which bring about a reduction in the unspecific bonds, the negative charges can in principle be distributed in the molecule. For example, it is possible to bind the negative charges in the form of a group described above with a pK _a of less than 5.0 (3.0) to a base-bearing basic unit, but it is also possible to bind them outside the sequence of base-containing basic units, e.g. . B. at one or both ends of the oligomers or to the nucleobase itself (eg DE-19712530). It has been found that in this case the negative charges advantageously have additional, non-base-bearing bases. scaffolding units. which contain a group with a pK of less than 5.0 (3.0) at one end of the base sequence, the label or the immobilizable group also being located on one or more of these additional basic unit units. In the case of peptide nucleic acids, it has proven to be extremely useful to condense further basic scaffold units from amino acids with a pK of less than 5.0 (3.0) already during the synthesis of the PNAs and to bind the labeling group to the additional basic scaffold units in a subsequent step. As a result, the additional basic framework units also act as a linker between the label and the base sequence specific for the nucleic acid to be detected. Those probes in which both negative charges are present within the base sequence and in the linker appear to be particularly advantageous.

1 shows some formulas of monomers according to the invention (1. 3, 4, 6 and 8) in protected form, as they can be used for oligomer synthesis, and the monomer units thus produced within a PNA (2, 5, 7 and 9) (Z: benzyloxycarbonyl, Bn: benzyl).

2 shows the influence of the salt content on the signal for a labeled DNA probe, a labeled probe according to the invention, a positively charged PNA probe and a probe with a negative charge only in the linker (outside the backbone).

The following examples illustrate the invention:

example 1

Synthesis of the monomers

Synthesis of the Boc-T-D-Glu (Bn) -OH PNA monomer

1. Synthesis of D-Glu (Bn) -0-allyl

9.64 g (29.6 mmol) of cesium carbonate (Fluka) and then are added with stirring to a solution of 10 g (29.6 mmol) of Boc-D-Glu (Bn) -OH (available from Novabiochem AG) in 100 ml of absolute DMF one 'Λ hour 7.16 g (59.2 mmol) allyl bromide (Fluka) and stirring continues for one% hour. The solid is then filtered off and the DMF is removed in vacuo. The residue is taken up in 400 ml of ethyl acetate and washed with 400 ml of saturated sodium hydrogen carbonate solution. Then the aqueous phase is extracted three times with 200 ml of ethyl acetate. The combined organic phases are then dried over Na _: SO. It is filtered off and rotated in. Now the residue is dissolved in 100 ml of benzene and mixed with 5.80 g (30.5 mmol) of p-toluenesulfonic acid. The mixture is boiled under reflux for 1 hour, then the solution is concentrated to a volume of about 20 ml and the product is crystallized by slowly adding diethyl ether. The solid is filtered off and recrystallized from methanol / diethyl ether. After drying under high vacuum 1 1.1 g (77%) of the product are obtained as p-toluenesulfonic acid salt in the form of colorless crystals.

2. Synthesis of Boc- (2-aminoethyl) -D-glutamic acid-γ-benzyl ester-α-allyl ester

8 g (16.4 mmol) of D-glutamic acid-γ-benzyl ester-α-allyl ester are shaken in a separating funnel with 400 ml of dichloromethane and saturated sodium bicarbonate solution. After the organic phase has been separated off, the aqueous phase is back-extracted three times with 200 ml of dichloromethane each time. The combined organic phases are dried over Na ₂ SO ₄ , filtered off and rotated in. The residue is dissolved in 60 ml of methanol and cooled in an ice bath. Then 3.14 g (19.7 mmol) of Boc-aminoacetaldehyde (produced according to KL Dueholm et al. Org. Prep. Proced. Int. 1993, 25, 457-461) are added with stirring and the mixture is stirred for one hour in an ice bath further. Then 1.1 ml of acetic acid and 0.55 g (8.75 mmol) of sodium cyanoborohydride are added. After ¹ Λ hour the methanol is removed in vacuo and the residue is taken up in 300 ml of ethyl acetate. The solution is mixed with 150 ml of a saturated sodium hydrogen carbonate solution and 150 ml Washed saline solution. The organic phase is then dried over Na ₄ SO ₄ , filtered off and evaporated. The residue is taken up in 120 ml of diethyl ether, cooled to -20 ° C. and mixed with the equivalent amount of 1.5 M anhydrous, ethereal HC1. Then add 120 ml of absolute hexane. The precipitated hydrochloride is filtered off. After recrystallization from methanol / diethyl ether / hexane, 7.15 g (88%) of colorless crystals are obtained.

3. Synthesis of Boc-T-D-Glu (Bn) -O-allyl

To a solution of 3.72 g (20.2 mmol) of thymine-1-acetic acid (produced according to KL Dueholm et al., J. Org. Chem. 1994, 59, 5767-5773) and 3.3 g (20.2 mmol) of 3,4-dihydro -3-hydroxy-4-oxo-l, 2,3-benzotriazine (Fluka) in 60 ml DMF, 4.37 g (21.2 mmol) DCC (Fluka) are added with stirring. After 1 hour, 5 g (10.1 mmol) of Boc- (2-aminoethyl) -D-glutamic acid-γ-benzyl ester-α-allyl ester hydrochloride and 1.75 ml (10.2 mmol) of Hünig's base are then added. The mixture is stirred at 40 ° C. overnight (approx. 15 h). The DMF is then stripped off in vacuo and the residue is taken up in 180 ml of ethyl acetate. The dicyclohexylurea formed is filtered off. The organic phase is then washed successively with 150 ml of saturated sodium bicarbonate solution, semi-saturated potassium hydrogen sulfate solution and and saturated sodium chloride solution, then over Na _; SO ₄ dried. It is filtered off and rotated in. The residue is purified on a silica gel column using ethyl acetate / hexane 2: 1 as the elution system. The product is then crystallized using ethyl acetate / diethyl ether. Yield: 3.6 g (57%).

4. Synthesis of Boc-T-D-Glu (Bn) -OH

37 mg (0.032 mmol) of tetrakistriphenylphosphine-palladium are added to a solution of 2 g (3.2 mmol) of Boc-TD-Glu (Bn) -O-allyl and 2.8 ml (32 mmol) of morpholine in 30 ml of absolute THF. After an hour, the THF is removed. The residue is taken up in 100 ml of ethyl acetate and washed with saturated sodium hydrogen carbonate solution, which was adjusted to pH 3 with potassium hydrogen sulfate solution. The aqueous phase is then back extracted 5 times with 50 ml of ethyl acetate. The combined organic phases are then washed with saturated sodium chloride solution and dried over Na 2 SO 4. It is filtered off and rotated in. After crystallization from ethyl acetate / hexane, 1.46 g (78%) of an almost colorless solid is obtained. 'H NMR (DMSO): COOH not visible. 1 1.27 (sb. IH. NH). 7.36-7.30 (m, 6H, Ph, H-C (6)), 6.99 (sb, IH. NH), 5.10 (2s. 2H. CH ₂ Ph), 4.61-4.49 (m. 2H. CH ₂ Thy) , 4.20-4.05 (m. IH, CH), 3.68-2.91 (t, tm 6H. 2x CH _: N, CH ₂ COO), 2.39-2.31 (m. 2H. CH ₂ ), 1.74 (s, 3H. CH ₃ ), 1.38, 1.35 (2s. 9H. CH ₃ (Boc))

Example 2

Synthesis of the oligomers

Synthesis of the PNA of the sequence:

Ru (bpy) ₃ (Glu), CCA C (T-Glu) A TAG G (T-Glu) G GGT CTT Gly

The synthesis was carried out on an ABI 433 A peptide synthesizer from Applied Biosystems with modified software (T. Koch et al., J. Peptide Res. 1997, 49, 80-88). The syntheses were carried out on a 5 μmol scale in a 3 ml reaction vessel, a smaller measuring loop (150 μl) was used. The monomer components (PNA components: Boc-T-OH, Boc-A (Z) -OH, Boc-C (Z) -OH and Boc-G (Z) -OH, available from Perseptive Biosystems, Boc-T-Glu (Bzl) -OH prepared according to Example 1; amino acid derivatives: Boc-Glu (OBzl) -OH, available from Novabiochem AG; Ru (ruthenium) (bpy) ₃ -COOH from Boehringer Mannheim) were in NMP dissolved and injected into individual cartridges (140 μl, 0.26M). The MBHA carrier material (Novabiochem AG) derivatized with Boc-Gly (Novabiochem AG) is filled into the 3 ml reaction vessel and attached to the synthesizer. Trifluoroacetic acid / m-cresol 95: 5 (2 x 180 sec); Bottle position 2) is used to split off the Boc protecting group. Two washing modules (dichloromethane and NMP module, each consisting of 5 consecutive washing steps) are located between the Boc split-off and the coupling module (dichloromethane = bottle position 9; NMP = bottle position 10). 140 μl 0.26 M monomer (36 μmol), 150 μl 0.21 M HATU (O- (7-azabenzotriazol-l-yl) -l, l, 3,3-tetra-methyluronium hexafluorophosphate) in NMP (32 μmol) and 150 μl 0.5 M diisopropylethylamine in NMP (75 μmol) are conveyed in each coupling step (diisopropylethylamine solution = bottle position 7; HATU solution = bottle position 8). The monomers are preactivated in the synthesis cartridge (1 min), then transferred to the reaction vessel. The coupling time is 10 min. the monomer concentration during the coupling is 0.08 M. After the coupling module there is a capping module with Acetic anhydride / NMP / pyridine 1:25:25 (1 min: bottle position 4). Each synthesis cycle is completed with an NMP wash module. After the last synthesis cycle, a dichloromethane wash module follows to dry the resin. The couplings were regularly checked by a qualitative Kaiser test.

The PNA is split off from the resin and the protective groups in a manual step outside the device in a special, lockable glass frit. The resin is first washed with trifluoroacetic acid, then shaken for 2 hours with 2 ml of trifluoromethanesulfonic acid / trifluoroacetic acid / m-cresol 2: 8: 1. The separation solution is sucked into a centrifuge glass. It is washed with 1 ml of trifluoroacetic acid and the PNA is precipitated with diethyl ether (approx. 10 ml). The precipitate is separated from the supernatant solution after centrifugation. Then it is washed twice with diethyl ether.

The PNA was analyzed by means of an analytical RP18-HPLC (DeltaPak. Waters, 5 μ, 125 × 4 mm) with a water / acetonitrile / 0.1% trifluoroacetic acid gradient at 60 ° C.

The PNA was purified by preparative RP18-HPLC (Polygosil C-18 / Macherey-Nagel, 5 μ, 250 x 20 mm) with a water / acetonitrile / 0.1% trifluoroacetic acid gradient at 60 ° C. until chromatographic uniformity.

The analysis of the purified PNA is carried out with MALDITOF-MS.

Example 3

HGV amplificate detection with differently modified detection probes

A comparison of a detection probe according to the invention with unmodified or otherwise modified PNA-Ru probes using an identical sequence illustrates the advantages of the invention. A DNA probe was used as a reference; the experiment was carried out using the example of HGV. a) amplification

To produce the tested PCR products (HGV or HBV), which served as samples in the example, plasmids were used which were amplified by means of PCR. The following primers were used to prepare the specific amplificates:

HGV primer sequences: coding-strand:

5 '-CGG CC A AAA GGT GGT GGA TG-3' 20-mer (SEQ.ID.NO 1) non-coding beach:

5'-Bi-CGA CGA GCC TGA CGT CGG G-3 '19-mer (SEQ.ID.NO. 2)

HBV primer sequences: coding-strand:

5'-GGA GTG TGG ATT CGC ACT-3 '18-mer (SEQ.ID.NO. 3) non-coding beach:

5'-TGA GAT CTT CTG CGA CGC-3 '18-mer (SEQ.ID.NO.4)

The following reaction approach was chosen for the PCR for both parameters:

2.5 U Taq polymerase

10 mM Tris / HCl. pH 8.3

50mM KCl, 1.5mM MgCL

0.1 mg / ml gelatin

200 nM dCTP, dATP, dGTP, 600 mM dUTP

200 nM coding beach primer

200 nM non-coding beach primer. Labeled biotin

10 ng plasmid pJCP (HGV: nucleotides 1-550 cloned in pGEM3z; Stratagene: HBV:

Nucleotides 50-2670 cloned in pUC 18)

The amplification was carried out with the following temperature profile on a Perkin Elmer Cycler 9600:

35 cycles: 15 sec 94 ° C

30 sec 55 ° C

30 sec 72 ° C hold 4 ° C

In order to stabilize the amplificate, the PCR product was purified after the end of the reaction using the High pure PCR template purification kit (BM Order No. 1 796 828). The PCR product in the specified dilutions was used to evaluate the various PNA-Ru probes. b) detection

The detection was carried out with the aid of electrochemilinescence (ECL) on an Elecsys® 1010 (Boehringer Mannheim GmbH). The reaction temperature for all steps is 37 ° C. 10 μl of sample are first mixed with 35 μl of denaturing reagent and incubated for 5 minutes. Then 120 μl of the probe solution (50 ng / ml in hybridization buffer) are added and incubated for 20 min. After the hybridization has ended, 35 μl of streptavidin-coated magnetic microparticles are added to the reaction mixture and incubated again for 10 minutes. The hybrids (bio-labeled HGV strand with Ru-labeled detection probe) are bound to the solid phase (microparticles). After the incubation has ended, the samples are transferred to the measuring cell and the microparticles are bound there by a magnet. The chemiluminescence then takes place in the measuring cell. When catalysed by TPA, the Ru complex emits flashes of light. the number of which are determined in the measuring cell. The chemistry for electrocheminoluminescence is in J. Electrochem. Soc. 137: 3127-3131.

Detection reagents:

1. Denaturing reagent. pH> 12

Hybridization solution, pH 6.5:

The following ruthenium bispyride complex-labeled probes were used as detection probes:

A. DNA probe:

Ru- CCA CTA TAG GTG GGT CTT Id.No. 55 (SEQ.ID.NO. 5)

B: PNA probes:

Ru Glu ₂ CCA CT (TGlu) A TAG GT (TGlu) G GGT CTT Gly Id.No. 506

Ru Gly ₃ CCA CTA TAG GTG GGT CTT Gly Id No. 487

Ru Glu, CCA CTA TAG GTG GGT CTT Glu Id.No. 493

Ru Gly ₃ CCA CT (TLys) A TAG GT (TLys) G GGT CTT Gly Id.No. 494

The probes were tested in the experiment described above, the result is shown in Table 4. The first part (4a) shows the mean value of the signals from a double determination, the second (4b) shows the ratio of the signal to the background signal.

The key findings are:

• The integration of TGlu in the backbone leads to a significant reduction in the background and is comparable to the reference (DNA probe)

• The modification leads to an improvement in the sensitivity of the PNA probe, the sensitivity is comparable to the reference. This can be seen clearly in Tab. 4b.

• In contrast to the reference, the sensitivity does not deteriorate when the salt concentration in the hybridization buffer is low. The competition reaction between unlabeled strand from the PCR reaction with the detection probe becomes lower Salt concentration shifted towards the probe, which enables a large dynamic measuring range with very good sensitivity.

• The modified probe according to the invention allows a particularly large dynamic measuring range to be covered, which is particularly important for quantitative detection

• With the unmodified PNA probes or with the positively charged PNA probe, the background increases with decreasing salt concentration

Table 4

4a Ident No. 55 506 493 494 487

Salt Concentration 50 mM 625 mM 50 mM 625 mM 50 mM 625 mM 50 mM 625 mM 50 mM 625 M

sample

HGV PCR product 1: 5 424834 666051 1184377 332695 1570831 945988 1084935 247646 1687111 463255

1:25 260813 728338 893928 427947 1237424 1037714 968912 355618 1325930 672337

1: 125 100840 338543 327337 238796 423002 358771 324117 182561 386787 295422

1: 625 5651 17490 19520 14311 115541 27823 69209 33119 39449 19414

1: 3125 4609 13937 15148 12732 103158 17725 60731 26740 19343 5809

1: 15625 1314 2528 3475 2784 100192 16068 58124 25929 17971 4870

H-O 879 890 1408 1239 93965 14343 59385 24997 16579 4172

Signal / noise ratio (signal / signal H20)

4b Ident No. 55 506 493 494 487

Salt concentration sample 50 M 525 mM 50 mM 625 mM 50 mM 625 mM 50 mM 625 mM 50 mM 525 mM

HGV PCR product 1: 5 483.31 748.37 841.18 268.52 16.72 65.95 18.27 9.91 101.76 111.04

1:25 296.72 818.36 634.89 345.40 13.17 72.35 16.32 14.23 79.98 161.15

1: 125 114.72 380.38 232.48 192.73 4.50 25.01 5.46 7.30 23.33 70.81

1: 625 6.43 19.65 13.86 11.55 1.23 1.94 1.17 1.32 2.38 4.65

1: 3125 5.24 15.66 10.76 10.28 1.10 1.24 1.02 1.07 1.17 1.39

1: 15625 1.49 2.84 2.47 2.25 1.07 1.12 0.98 1.04 1.04 1.07

H ₂ 0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Example 4

Reduction of the blank value and the non-specific cross-reactivity in ECL measurements

The implementation of the experiment is described in Example 3. A length standard marked with biotin (corresponding to Cat.No. 1062590, only 5 'biotin marked) was used as sample material. The concentration of the stock solution: 1 μl corresponds to 250 ng DNA.

The following detection probes were used:

A. DNA probe:

Ru- CCA CTA TAG GTG GGT CTT (SEQ.ID.NO. 5) Id.No. 55

B: PNA probes:

Ru Glu ₂ CCA CT (TGlu) A TAG GT (TGlu) G GGT CTT Gly Id.No. 506

Ru Gly ₃ TAT AGG TGG GTC TT Gly Id No. 484

Ru Gly ₃ ACT ATA GGT GGG TCT T Gly Id No. 486

The result is shown in Table 5. 5a shows the mean value of the signals from a double determination, FIG. 5b shows the ratio of the signal to the background signal. The key findings are:

• Nonspecific hybridization reactions can only be observed with the two unmodified PNA probes, but not with the probe modified according to the invention or with the reference.

• The strength of the non-specific reactions increases with the length of the unmodified probes (484: 14-mer; 486: 16-mer).

Table 5 Probe 55 506 484 486

5a salt OmM 50 mM 625 mM OmM 50 mM 625 mM 50 mM 625 mM 50 mM 625 mM

sample

HGV PCR product 1: 5 246843 424834 666051 1211085 1184377 332695 1331089 823696 2802342 1274087

1:25 149673 260813 728338 795203 893928 427947 787229 873993 3000544 1790686

1: 125 64415 100840 338543 308401 327337 238796 293682 337931 922030 694632

1: 625 3850 5651 17490 20053 19520 14311 15307 19104 37393 30758

1: 3125 3028 4609 13937 13965 15148 12732 4324 6090 9437 5436

1: 15625 1188 1314 2528 2844 3475 2784 3944 5204 7423 3864

H ₂ 0 880 879 890 1204 1408 1239 3084 4432 5607 1925

HGV Primer 871 852 876 2186 2622 1494

Length marker VI 1: 100 739 743 900 1075 1266 1836 9225 10845 25825 7712

1:20 774 782 878 1462 1865 1344 22773 29318 87218 29046

1:10 811 809 867 1307 1524 1288 38744 46311 140360

HBV PCR product 1: 5 834 856 904 921 1123 1225

Probe 55 506 484 486

5b salt OmM! 50mM (325mM OmM! 50mM (525mM 50mM f 325mM 50mM (325mM

sample

HGV PCR product 1: 5 280.5 483.3 748.4 1005.9 841.2 268.5 431.2 185.9 499.8 661.9

1:25 170.1 296.7 818.4 660.5 634.9 345.4 255.0 197.2 535.1 930.2

1: 125 73.2 114.7 380.4 256.1 232.5 192.7 95.1 76.2 164.4 360.8

1: 625 4.4 6.4 19.7 16.7 13.9 11.6 5.0 4.3 6.7 16.0

1: 3125 3.4 5.2 15.7 11.6 10.8 10.3 1.4 1.4 1J 2.8

1: 15625 1.4 1.5 2.8 2.4 2.5 2.2 1.3 1.2 1.3 2.0

H ₂ 0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

HGV Primer 1.0 1.0 1.0 1.8 1.9 1.2

Length marker VI 1: 100 0.8 0.8 1.0 0.9 0.9 1.5 3.0 2.4 4.6 4.0

1:20 0.9 0.9 1.0 1.2 1.3 1.1 7.4 6.6 15.6 15.1

1:10 0.9 0.9 1.0 1.1 1.1 1.0 12.6 10.4 25.0

HBV PCR product 1: 5 0.9 1.0 1.0 0.8 0.8 1.0

Example 5

Influence of salinity on the sensitivity of nucleic acid detection

Analogously to example 3, HGV was determined with the aid of a DNA probe (diagram 1), a positively charged PNA probe (diagram 3) and two negatively charged probes (diagrams 2 and 4). The salt content (0; 50; 150; 200; 300; 625 mM) was varied. The measurement results are shown graphically in FIG. The results are discussed in the description.

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Boehringer Mannheim GmbH

(B) STREET: Sandhoferstr. 1 16

(C) LOCATION: Mannheim

(E) COUNTRY: DE

(F) POSTAL NUMBER: 68305

(G) TELEPHONE: 0621 759 4348 (H) TELEFAX: 0621 759 4457

(ii) NAME OF THE INVENTION: Modified nuclein analogs (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CGGCCAAAAG GTGGTGGATG 20

(2) INFORMATION ON SEQ ID NO: 2: (i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CGACGAGCCT GACGTCGGG 19

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GGAGTGTGGA TTCGCACT 18

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGAGATCTTC TGCGACGC 18 (2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCACTATAGG TGGGTCTT 18

Claims

Expectations

1. Molecule hybridizing with nucleic acids with a backbone of linearly linked monomer units, at least one of the monomer units being a non-nucleoside unit which contains an acid function with a pK of less than 3.0.

2. Molecule according to claim 1 containing at least one non-nucleoside monomer unit of the formula I.

[!]

(I)

wonn

L is selected from the group hydrogen, hydroxyl, (C, - C ₄ ) - alkanoyl, nucleobase-binding group, (C ₆ -C ₁₄ ) aromatics. Heterocycles with nitrogen, oxygen or / and sulfur atoms, intercalators and reporter groups,

A is a spacer with a length of 1 to 3 atoms,

x is a number between 0 and 3,

B is a basic unit with a length of between 4 and 8 atoms,

R is a group containing an acid function with a pK_ of less than 3.0

mean.

3. Molecule according to one of the preceding claims, characterized in that L is bonded via A to a nitrogen atom in B.

4. Molecule according to claim 3, characterized in that R is not bound to the nitrogen atom.

5. Molecule according to one of the preceding claims, characterized in that the monomer unit has the formula II,

wonn

E 'and F' independently of one another CO, CS, SO, SO, or NR ¹ ,

where Rl is selected from the group hydrogen and (C, -C ₃ ) alkyl

N is an amine nitrogen atom,

C (CR ² R ³ ) _n or CR ² R ³ CR ⁴ R ⁵

in which

R ² , R ³ , R ⁴ and R ^{5 are} independently selected from the group hydrogen, C, -C ₆ - (substituted or unsubstituted) alkyl, hydroxyl, carboxyl and a group containing the acid function, and

n is a number between 1 and 4, and

D (CR ² R ³ ) _n or CR ² R ³ CR ⁴ R ⁵

in which

R ² , R \ R ⁴ and R ^{5 are} independently selected from the group hydrogen, C, -C ₆ - (substituted or unsubstituted) alkyl, hydroxyl, carboxyl and a group containing the acid function, and

n is a number between 1 and 4, mean.

6. Molecule according to claim 5, characterized in that D is derived from serine or a homologue of serine.

7. Molecule according to one of the preceding claims, characterized in that the acid function is selected from the groups of sulfonic acids, phosphonic acids or phosphoric acids.

8. Molecule according to one of the preceding claims, characterized in that the acid function is bound via a spacer A 'of between 1 and 20 atoms in length to linearly consecutive atoms of the basic structure.

9. Molecule according to claim 8, characterized in that the spacer A 'has the formula III

- (- (- A'-) _a - (- A ² -) _b - (- A ³ -) c-) d- (HI)

has in the

A ¹ oxygen, NR ⁶ or sulfur,

A ^{2 is} a substituted or unsubstituted alkylene. Alkynylene or

Alkenylene rest.

A ³ oxygen, sulfur or NR ⁶ ,

R ^{6 is} independently H or alkyl or acetyl,

a and c independently of one another a number from 0 to 2.

b is a number from 1 to 10, and

d is a number from 1 to 4

mean.

10. Molecule of formula IV

(IV)

in the

L is selected from the group hydrogen, hydroxyl, (C, -C ₄ ) -

Alkanoyl, nucleobase-binding group in protected or unprotected form, (C ₆ -C, ₄ ) aromatics, heterocycles with nitrogen, oxygen or / and sulfur atoms, intercalators and reporter groups,

A is a spacer with a length of between 1 and 3 atoms.

x is a number between 0 and 3,

E and F independently of one another COOX, CSOX, SO _: X, SO ₃ X or NR ¹ Y,

in which

R ^{1 is} selected from the group hydrogen and (C, -C ₃ ) alkyl,

X is selected from the group consisting of hydrogen, the protective groups and the activation groups.

Y is selected from the group hydrogen and the protective groups,

N is an amine nitrogen atom. and

in which

R \ R \ R ⁴ and R ^{5 are} independently selected from the group hydrogen. C, -C ₆ - (Substituted or unsubstituted) alkyl, hydroxyl, carboxyl and an acid function with a pK. of less than 3.0 group in protected or unprotected form, and n is a number between 1 and 4.

11. A method for detecting the presence or absence of a nucleic acid in a sample, which is suspected to contain one or more other nucleic acids whose base sequence differs from the base sequence of the nucleic acid to be detected in one or more positions, comprising the steps

Contacting a probe which contains a base sequence which is complementary to the base sequence to be detected, but not a base sequence of the other nucleic acids, and

Determining whether binding of the probe to a base sequence complementary to the base sequence has taken place,

characterized in that a molecule is used as the probe, in which less than 80%, but at least one of the bases is bound to negatively charged basic framework units.

12. The method according to claim 1 1, characterized in that the non-nucleoside basic unit at the different position differs from at least one of the two adjacent basic units.

13. The method according to any one of claims 1 1 and 12, characterized in that the different position is at the end of the base sequence.

14. The method according to any one of claims 11 to 13, characterized in that the probe bears a label.

15. The method according to claim 14, characterized in that the label is a ruthenium bispyridyl complex.

16. Use of a method according to any one of claims 11 to 13 for the detection of mutations. Alleles or polymorphisms.

17. Use of a method according to any one of claims 1 1 to 13 for typing or subtyping microorganisms.

18. Use of negative charges in probes which contain at least one non-nucleoside basic unit to avoid unspecific binding of the probes to base sequences which are less than 50% complementary to the probe or to solid supports.

19. Use of acid functions to increase the selectivity of probes in binding reactions.

20. Use of molecules hybridizing with nucleic acids, in which at least one of the bases is bound to a non-nucleosidically negatively charged basic unit, as a primer for polymerase-catalyzed extension with the aid of mononucleoside triphosphates.

21. Use of nucleic acid-binding molecules in which at least one of the bases is bound to a non-nucleosidically negatively charged basic unit for transfection in cells with the aid of transfection reagents.