ES2352509T3 - NEW MARKING STRATEGIES FOR THE SENSITIVE DETECTION OF ANALYTES. - Google Patents

Abstract

A method for detecting an analyte, selected from nucleic acids, in a sample, comprising the steps: (i) providing a sample; (ii) contacting the sample with a functionalized compound comprising at least one functional group selected from alkyne and azide groups that is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between the azide and alkyne groups that form a 1,2,3-triazole ring, and in which the functional group is attached to a nucleobase of a nucleoside or nucleotide compound or an oligomer or polymer thereof, under conditions in which said compound forms an association product with the analyte to be detected, or its complement, (iii) contacting the association product with a second reaction partner for said Click reaction, which comprises the complementary azide or alkyne group, under conditions in which a Click reaction occurs between the first and second reaction couple, in which the second reaction couple further comprises a marker group or a precursor group of the marker, (iv) if necessary, convert the marker precursor groups into marker groups, and (v) detect said marker groups.

Description

The present invention relates to methods and the use of reagents for detecting analytes, for example nucleic acids. The new methods and reagents allow simple and sensitive detection, even in complex biological samples.

Background of the Invention

Rapid analysis of genetic material for specific target sequences, for example to determine the presence of single nucleotide polymorphisms, the presence of a certain gene, for example a resistance gene, or 10 mRNA, requires new easy-to-use tools. Use, effective and reliable. The main problem is the need to detect the DNA or RNA of interest directly in small biological samples, such as the patient's blood or plants. These provide the analyte only in tiny amounts. In order to achieve the required sensitivity, an amplification step is usually required, in which the nucleic acid analyte is amplified before the analysis, or a detection method is used, in which the tiny detection signal is amplified, directly 15 obtained from the DNA / RNA analyte.

Methods for nucleic acid analyte amplification include PCR and other nucleic acid amplification protocols. PCR amplification has the main advantage that, within a set of different strands of DNA obtained from the biological material, only the DNA sequence of interest is amplified. This is the basis for reliable analysis of individual genes in complex biological samples. However, amplification by PCR 20 requires complex procedural steps that, in some cases, are too inconvenient and expensive. Amplification of the detection signal can be achieved by attaching an enzyme, such as horseradish peroxidase, to the analyte, which continuously converts a given substrate into a colored product.

Another way to amplify the detection signal is the metallization of DNA. A single metal particle bound to DNA / RNA (the nucleus) catalyzes the deposition of more and more metal, which catalyzes the additional deposition of metal [1-9]. 25 The signal induced by metallic deposition grows exponentially accordingly. The metallic deposition can be detected electrically, if the analyte is placed between the electrodes, or optically (for example, with the eye), because the deposited metal gives rise to a black spot, for example on paper, gel, or in the test tube. In principle, metal deposition is the most sensitive detection method because a small metal cluster (core) is sufficient to start the reaction. However, in practice, the sensitivity of the method is limited by non-specific metal nucleation, for example through impurities in a spatial neighborhood close to the analyte, for example in the electrodes, in the gel, or in the role it has. The analyte In fact, non-specific metallic deposition is the main reason why DNA staining with silver is not commonly used in oligonucleotide analytics. Silver staining is further complicated by the fact that DNA is unable to build the initiation nucleus itself. It requires a previous modification. The method of choice currently is the reaction of DNA with glutaraldehyde, which covalently binds to DNA by unspecific binding of the sequence to primary amine groups in the nucleobase [7]. This adduct, if treated with a silver salt, reduces the Ag + of the salt to atomic silver, which, while attached to DNA, functions as the required nucleus. Subsequent treatment of nucleated DNA with silver salts and reducing agents initiates exponential metal deposition. Another possibility is to exchange the counterions in the DNA strand for Ag +, which is subsequently reduced to give Ag0 nucleation sites. The main disadvantage is that glutaraldehyde also reacts with impurities or other chemical species close to the analyte, which again induces non-specific deposition of silver.

Metal clusters such as Au, Pd particles or Pt complexes bound to DNA also function as nucleation sites for subsequent deposition of metal to the construction of conducting wires [1-9]. Here, the clusters bind to reactive groups that form a covalent bond with DNA, or units that only intercalate or otherwise bind to DNA / RNA. All these methods mark all the DNA in a biological sample, and therefore do not allow the specific labeling of a sequence and therefore the specific analysis of a sequence of a target DNA, such as a single gene in a complex biological sample.

[8] describes the preparation of labeled DNA domains by means of a telomerase-mediated incorporation of amine modified nucleoside triphosphates into a modified telomeric repeat initiated by primers. 50 Telomeres containing amines are functionalized with N-succinimidyl esters of activated gold nanoparticles, to produce DNA strands with gold nanoparticles. The elongation of these nanoparticle sites by subsequent metal deposition along the DNA actually causes rapid growth of the metal clusters until the construction of tempered molecular nanowires of DNA. However, the effectiveness of incorporating the modified triphosphate with amines at the growing telomeric end is low, and requires doping of the triphosphate, which results in a distribution of the nucleation sites. Thus, the procedure does not allow specific sequence marking.

Recently, site specific DNA labeling has been attempted via a complex lithographic method [4]. According to this method, a partial protection of the DNA molecules is effected by the binding of RecA. The unprotected DNA sequences are then treated with glutaraldehyde, which marks these sequences for metallization. The specific reduction of the site of silver ions by DNA-bound aldehyde functions results in the formation of a wire throughout these regions. However, specific labeling of nucleic acid molecule sequences in a complex biological sample is not possible.

HC Kolb et al. (HC Kolb, “The growing impact of click chemistry on drug discovery,” Drug Discovery Today, vol. 8, no. 24, December 15, 2003, pages 1128-1137) shows, on page 1135, in scheme 4c, a universal direct sequencing primer that has a Click functional group attached. In this case, however, the Click functional group is linked by a linker to the phosphate group at the 5 'end of the primer, while, according to the present invention, the Click functional group is attached to a nucleobase. The difference is obvious when the description of TS Seo et al. (TS Seo et al., "Click chemistry to construct fluorescent oligonucleotides for DNA sequencing",
J. Org. Chem., Vol. 68, nº 2, December 21, 2002, pages 609-612). This document is cited 10 as reference [60] in Figure 4c by HC Kolb et al. TS Seo et al. show, in scheme 1, on page 610, the preparation of azide-modified nucleic acid 2 by reacting the commercially available universal direct sequencing primer M13-40 with succidinyl 5-azidovalerate. The azido labeled DNA mentioned by TS Seo et al. and HC Kolb et al. it thus possesses an azido group in the phosphate group at the 5 'end, but not in a nucleobase. fifteen

Although these documents demonstrate interest in new DNA labeling strategies, the complicated procedures involved in order to achieve labeling prevent these systems from being used for any real application. In particular, these methods are unable to selectively label DNA or RNA sequences of interest directly in a crude biological sample.

Thus, it was an object of the present invention to provide new methods and reagents that allow a simple, efficient and specific detection of analytes, particularly nucleic acids in complex biological samples.

Summary of the Invention

The present invention relates to reagent methods and kits for detecting an analyte, for example a nucleic acid in a sample, which involve the use of a functionalized compound by Click that forms a product of association with the analyte to be detected. The specific introduction of marker group sequences is effected by reacting the functionalized compound by Click with a suitable reaction partner comprising marker groups or marker precursor groups.

An embodiment of this aspect relates to a method for detecting an analyte, selected from nucleic acids, in a sample, comprising the steps of:

(i) provide a sample; 30

(ii) contacting the sample with a functionalized compound comprising at least one functional group selected from alkyne and azide groups that is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between the azide and alkyne groups that form a 1,2,3-triazole ring, and in which the functional group is attached to a nucleobase of a nucleoside or nucleotide compound or an oligomer or polymer thereof, under conditions in which which said compound forms an association product with the analyte to be detected,

(iii) contacting the association product with a second reaction partner for a Click reaction comprising the complementary azide or alkyne group, under conditions where a Click reaction occurs between the first and second reaction couple, wherein the second reaction partner further comprises a marker group or a marker precursor group,

(iv) if necessary, convert the marker precursor groups into marker groups, and

(v) detect said marker groups.

A further embodiment of this aspect relates to a reagent kit for detecting an analyte in a sample, comprising:

(a) a functionalized compound comprising

(i) at least one Click functional group, selected from alkyne and azide groups, which is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between azide and alkyne groups which forms a 1,2,3-triazole ring, and

(ii) a building block of a nucleic acid or nucleic acid analogue, in which the functional group is linked to a nucleobase,

(b) a second reaction pair for a Click reaction, comprising the complementary azide or alkyne group, wherein said second reaction pair further comprises a marker group or a marker precursor group, and

(c) optionally a marker forming reagent, capable of converting the marker precursor groups into marker groups. 5

Still a further embodiment of this aspect refers to the use of a compound of formula (II):

C-S-N

wherein C is a Click functional group selected from alkyne and azide groups that is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between azide and alkyne groups forming a 1,2,3-triazole ring, 10

S is a spacer, and

N is a building block of nucleic acid or a nucleic acid analog, such as a nucleoside or nucleotide compound, in which the Click functional group binds to a nucleobase, in a method to detect a nucleic acid analyte in a sample,

wherein the compound of formula (II) forms a product of association with the analyte to be detected. fifteen

A further embodiment of this aspect relates to a nucleic acid molecule or a nucleic acid analog having at least one compound of formula (II) incorporated therein as defined above, in which the functional group is an alkyne group linked to positions 5 and 6, preferably position 5, of a pyrimidine nucleobase, or positions 7 and 8, preferably position 7, of a purine nucleobase, and in which the nucleic acid or acid analog molecule Nucleic is capable of hybridizing with a complementary nucleic acid. twenty

A further embodiment of this aspect relates to a method for synthesizing a nucleic acid molecule or a nucleic acid analog that is capable of hybridizing to a complementary nucleic acid, which comprises incorporating a nucleotide building block or a nucleotide analog comprising a compound (II) as defined above, in which the functional group is an alkyne group attached to positions 5 and 6, preferably to position 5, of a pyrimidine nucleobase, or to positions 7 and 8, preferably position 7, of a purine nucleobase in a nucleic acid molecule or a nucleic acid analog.

This method may further comprise contacting the nucleic acid molecule or a nucleic acid analog with a second reaction partner of compound (II) and carrying out a Click reaction between the first and second reaction partners, said said being Click reaction a cycloaddition reaction (3 + 2) between azide and alkyne groups that forms a 1,2,3-triazole ring. 30

A further embodiment of this aspect relates to an association product of the nucleic acid molecule or nucleic acid analog as defined above with an analyte.

A further embodiment of this aspect relates to a compound, obtainable as the reaction product of the method for synthesizing a nucleic acid molecule or a nucleic acid analog.

A further embodiment of this aspect relates to an association product of the compound as defined above with a complementary nucleic acid.

The present invention allows a very sensitive detection of an analyte, for example nucleic acids or proteins that bind to nucleic acids, in biological samples, for example clinical samples, environmental samples or agricultural samples. Preferred applications include, but are not limited to, the detection of genetic variability, for example single nucleotide polymorphisms (SNPs), resistance to pesticides or medications, tolerances or intolerances, genotyping, for example the detection of species or strains of organisms, the detection of genetically modified organisms or strains, or the detection of pathogens or pests, and the diagnosis of diseases, for example genetic diseases, allergic diseases, autoimmune diseases or infectious diseases. A further preferred application is the detection of nucleic acids in samples for the protection of trademarks, in which products such as agricultural products, food products, or valuable goods and / or the packages of these products 45 are encoded with specific information of the product, for example, but not limited to, production site, production date, distributor, etc., and in which this information is detected with the methods as described above.

Detailed Description of Preferred Embodiments

The invention provides methods and reagents that allow specific labeling of analytes with marker groups, for example with reactive aldehyde groups, in a complex sample. In a preferred embodiment, metal deposition, for example silver deposition, can be performed specifically on the aldehyde groups associated with the nucleic acid to be detected. In a further preferred embodiment, the metallic deposition, for example the deposition of silver, can be carried out specifically by transferring energy from photosensitizing groups to a photosensitive medium, for example photographic paper. Due to the specific marking procedure, the fund is strongly reduced, and greater sensitivities and reliability are obtained.

The present invention comprises the detection of an analyte. The detection can be a qualitative detection, for example the determination of the presence or absence of an analyte, for example a specific nucleic acid sequence, in the sample to be analyzed. However, the invention also allows quantitative detection of an analyte, for example a nucleic acid sequence, in the sample to be analyzed. Qualitative and / or quantitative detection may comprise the determination of marker groups according to methods known in the art.

The analyte to be detected is preferably selected from nucleic acids and molecules that bind to nucleosides, nucleotides or nucleic acids, for example proteins that bind to nucleosides, nucleotides or nucleic acids. More preferably, the analyte is a nucleic acid, for example any type of nucleic acid that can be detected according to known techniques, particularly hybridization techniques. For example, nucleic acid analytes can be selected from DNA, for example double-stranded or single-stranded DNA, RNA, or DNA-RNA hybrids. Particular examples of nucleic acid analytes are genomic DNA, mRNA or products derived therefrom, for example cDNA. fifteen

The method of the invention can be carried out according to any known assay format that is suitable for the detection of analytes, particularly nucleic acid analytes, in a sample. For example, the method may involve the detection of immobilized analytes on solid surfaces such as membranes, for example Southern or Northern blots, chips, matrices or particles such as beads. In addition, the detection can be carried out in gels, for example after electrophoretic separation of the sample in gels, for example agarose or polyacrylamide gels. The method may involve the detection of individual analytes, or the parallel detection of a plurality of analytes, for example in a chip or microarray format.

In a preferred embodiment, the detection involves irradiating a photosensitive medium in the presence of a sample containing an indicative association product for an analyte, in which the association product comprises photosensitizing groups capable of effecting a transfer of energy to the photosensitive medium in the that groups 25 markers are formed in the middle.

The sample can be any sample that may contain the analyte to be detected. For example, the sample may be a biological sample, such as an agricultural sample, for example a sample comprising plant material and / or material associated with the site where the plants grow, where plant materials are stored or processed. On the other hand, the sample may also be a clinical sample, such as a tissue sample or a sample of body fluid such as blood, serum, plasma, etc., particularly of human origin. Other types of samples include, but are not limited to, environmental samples, soil samples, food samples, forensic samples, or samples of valuable goods that are tested for brand protection.

Due to its high sensitivity, the method of the present invention is suitable for directly detecting analytes without amplification. According to the invention, even tiny amounts of analytes, for example of nucleic acids, for example 0.1 ng or less, preferably 0.01 ng or less, more preferably 1 pg or less, can still be determined even without amplification. more preferably 0.1 pg or less, even more preferably 0.01 pg or less, and most preferably 0.001 pg or less. High sensitivity can be obtained by incorporating multiple modified nucleotides into a nucleic acid molecule using unprotected aldehyde groups and / or using optimized staining techniques. For example, the detection of an analyte, for example a gene, in a biological sample, can be carried out by a combination of Southern blotting and the method of the invention. However, it should be noted that the method of the present invention also allows the detection of nucleic acids combined with an amplification step, which can be carried out according to known protocols such as PCR or its modifications, such as asymmetric PCR, PCR in real time, reverse transcription PCR, etc., or other amplification protocols such as LCR. Four. Five

In a preferred embodiment of the invention, a sequence specific analyte detection is carried out, in which for example a nucleic acid is distinguished that has a specific sequence from other nucleic acid sequences in the sample, or a polypeptide is distinguished capable of binding to a specific nucleic acid sequence of other polypeptides in the sample. Such sequence-specific detection preferably comprises a sequence-specific hybridization reaction whereby the nucleic acid sequence to be detected is associated with a compound having a marker group or a group of a marker precursor. However, it should be noted that the present invention also allows the detection of non-sequence specific nucleic acids, for example the detection of any nucleic acids present in a sample.

In order to identify the analyte to be detected, the sample can be contacted with a functionalized compound by Click, under conditions in which an association product with the analyte is formed, for example a nucleic acid.

The functionalized compound may comprise a single functional group, or a plurality of functional groups. For example, a functionalized compound can be coupled to a dendrimeric moiety comprising a plurality, for example 2, 3, 4, 5, 6, 7, 8 or more functional groups as indicated above. Dendrimeric moieties can be synthesized by known techniques. Preferably, dendrimeric moieties are synthesized via Click reactions, for example as described in Example 5.

The functional group binds to a compound that is capable of forming an association product with the analyte. The compound may be a nucleoside or nucleotide compound, for example a nucleoside or nucleoside analog or a nucleotide or nucleotide analog or an oligomer or polymer comprising at least one functionalized compound, for example a nucleic acid or nucleic acid analog. A nucleoside or nucleotide compound is a nucleoside or nucleoside analog or a nucleotide or nucleotide analog capable of being incorporated into nucleic acids or nucleic acid analogs, for example by chemical or enzymatic methods. The resulting nucleic acid or nucleic acid analog should be capable of forming association products, for example nucleic acid hybrids, with the analyte. Preferably, the compound comprises a base moiety, for example a nucleobase, or other heterocyclic base moiety capable of forming base pairs with a nucleobase, and a main chain moiety, for example comprising a sugar moiety and optionally a phosphate moiety in nucleosides or nucleotides or a different main chain residue in nucleoside or nucleotide analogs.

In Figure 1, preferred examples of functional nucleoside compounds are shown, in which the nucleobase is 7 7-dN-G, C, 7-dN-A or T and R is a functional group.

Preferably, the functional group is linked to a base moiety, for example a nucleobase. The functional group, however, can also be linked to a main chain moiety, for example a sugar group, a phosphate group, or, in the case of nucleoside or nucleotide analogs, a modified sugar group, a modified phosphate group, or a remainder of the peptide main chain, etc. Preferably, the functional group is covalently linked to the compound via a direct bond or via a spacer. If the union is carried out via a spacer, the functional group can be linked to an aliphatic or cycloaliphatic group, an aromatic or heteroaromatic group, an alkene group and / or an alkyne group. More preferably, the functional group can be linked to aromatic or heteroaromatic groups, or to alkyne groups. Especially preferred aldehyde groups include aromatic and aliphatic aldehyde groups such as benzaldehyde, or aldehyde groups in aldoses such as trioses, tetroses, pentoses or hexoses such as glucose or mannose.

The functional group may also be a loop type group, that is, a group to introduce an aldehyde group, a protected aldehyde group or an aldehyde precursor group, or to introduce a photosensitizer group by reaction with a suitable reaction partner, it is that is, a compound comprising one of the above groups. The loop-like groups are selected from groups functionalized by Click, that is, groups that can react with a suitable reaction partner in a cycloaddition reaction in which a cyclic bond, for example heterocyclic, is formed between the functional group of Click and the reaction partner, and in which the reaction partner comprises an aldehyde or a protected aldehyde group or a photosensitizer group. An especially preferred example of such a Click reaction is a cycloaddition (3 + 2) between azide and alkyne groups, which results in the formation of 1,2,3-triazole rings. In this way, the aldehyde groups can be generated by carrying out a Click reaction of an azidic or alkyne loop group and a corresponding reaction partner, that is, a reaction partner comprising the complementary alkyl or azide group and additionally an aldehyde, a protected aldehyde group or an aldehyde precursor. In a further embodiment of the invention, the reaction partner of a Click functionalization, however, may also contain different marker groups or marker precursors such as fluorescence marker groups or photosensitizer groups. 40

An especially preferred embodiment of the Click reaction comprises a copper-loaded cycloaddition (3 + 2) between an azide and an alkyne group. The irreversible formation of 1,2,3-triazoles as a result of azide / alkyne cycloaddition is orthogonal, the chemical groups required are small (incorporation with minimal disruption of the biomolecule environment), and selective due to lack of azides and Alkynes found in nature.

 Four. Five

image 1

in which R1 and R2 are organic radicals.

Specific examples of compounds functionalized by Click, and the methods for preparing such compounds, are shown in Figures 3b, 9a-c and 10. Specific examples of reaction partners for compounds functionalized by Click are shown in Figures 11 and 12.

The group functionalized by Click is preferably linked to a nucleobase, which can be selected from purine and pyrimidine bases of natural and non-natural origin. Preferably, the nucleobases are selected from citidine, uracil, thymine, adenine, guanine, 7-desazaadenine, 7-desazaguanine, inosine and xanthine. The functional group is preferably attached to position 5 or 6, more preferably to position 5, of a pyrimidine nucleobase, or to position 7 or 8, more preferably to position 7 of a purine nucleobase, particularly if an enzymatic incorporation is desired. in a nucleic acid.

The functional group can be covalently linked to the compound, for example via a direct bond or a spacer, for example a spacer having a chain length of up to 20 atoms. The spacer can be a flexible spacer 5, for example an alkylene-based spacer, optionally containing heteroatoms such as O, S and / or N, or an at least partially rigid spacer, for example a spacer comprising at least one group rigid selected from alkene groups, alkyne groups, cyclic groups, particularly aromatic or heteroaromatic groups, but also cycloaliphatic groups and combinations thereof. If the functionalization compound comprises a functionalization group by Click which is the first reaction partner for a Click reaction, and is subsequently reacted with a second reaction couple for a Click reaction, a functional group bond is preferred. via a direct link, a flexible spacer or a partially rigid spacer, in which the flexible spacer could for example have a chain length of up to 6 atoms, more particularly up to 4 atoms, and in which a partially rigid spacer preferably has a chain length of up to 20 atoms, for example up to 10 atoms, and comprises at least one rigid group as defined above, particularly an alkyne group, and at least one flexible group, for example an alkylene group. On the other hand, if the functional group is an aldehyde group or a protected aldehyde group or an aldehyde precursor group, binding via a partially rigid spacer as defined above or an at least partially rigid spacer having a chain length is preferred. from 2 to 10 atoms. The structure of a spacer containing a rigid group, for example a partially rigid spacer, is preferably such that the rigid group is directly attached to the nucleobase. A preferred example of a particularly rigid spacer is shown in Fig. 15a and b.

The functionalized compound is capable of forming an association product with the analyte to be detected. On the other hand, the functionalized compound can be selected from compounds that can be incorporated into nucleic acids or nucleic acid analogs, ie nucleic acid building blocks or nucleic acid analogs. Such compounds are nucleotides or nucleotide analogs functionalized by Click. On the other hand, the functionalized compound 25 can be selected from nucleic acids or functionalized analogs by Click.

The term "nucleotide," according to the present invention, refers particularly to ribonucleotides, 2’-deoxyribonucleotides or 2’, 3’-dideoxyribonucleotides. Nucleotide analogs can be selected from sugar modified or modified backbone nucleotides, particularly from nucleotide analogs that can be enzymatically incorporated into nucleic acids. In preferred sugar modified nucleotides, the 2'-30 OH or H group of the ribose sugar is replaced by a group selected from OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is alkyl C1-C6, alkenyl or alkynyl, and halo is F, Cl, Br or I. The ribose itself can be substituted by other 5 or 6 membered carbocyclic or heterocyclic groups, such as a cyclopentane group or a cyclohexene group. In preferred modified main chain nucleotides, the phospho (tri) ester group may be substituted by a modified group, for example by a phosphorothioate group or an H-phosphonate group. Other preferred nucleotide analogs include building blocks for the synthesis of nucleic acid analogs such as morpholinic nucleic acids, peptide nucleic acids or blocked nucleic acids.

Click-functionalized nucleic acids can be oligonucleotides, for example nucleic acids that are up to 30 nucleotide building blocks (or nucleotide analogs), or polynucleotides that are more than 30 nucleotide building blocks (or nucleotide analogs) . Preferably, the nucleic acids and nucleic analogs are capable of specifically binding to the analyte, for example they are capable of hybridizing with a nucleic acid analyte under the test conditions. The minimum length is preferably 12 or more, preferably 14 nucleotide building blocks (or nucleotide analogs).

Nucleic acid or functionalized nucleic acid analog groups can be incorporated into nucleic acids by standard techniques for incorporation by chemical and / or enzymatic synthesis. Chemical synthesis can be carried out, for example, by standard phosphoramidite chemistry, using modified nucleoside phosphoramidites as building blocks in standard synthesis protocols. Other types of preferred building blocks for chemical synthesis include nucleosides modified by H-phosphonate or phosphorothrester.

On the other hand, modified nucleotides can be incorporated into nucleic acids by enzymatic methods. Surprisingly, it was found that nucleoside triphosphates functionalized by Click are accepted as enzyme substrates by enzymes that synthesize nucleic acids, such as DNA polymerases, RNA polymerases, reverse transcriptases or telomerases. For example, it was found that modified nucleoside triphosphates are accepted by DNA polymerases commonly used for extension and amplification protocols by primers, for example thermostable DNA polymerases such as Taq polymerase, Vent polymerase, Pfx 55 polymerase, Pwo polymerase or Therminator polymerase, as described in Example 7. Enzymes accept modified triphosphates without loss of fidelity, and allow the incorporation of molds into nucleic acids such as DNA and RNA.

The method of the present invention provides various embodiments of analyte detection. For example, functionalized nucleic acid building blocks, for example nucleotides or nucleotide analogs, may be provided, together with appropriate enzymes, which are enzymatically incorporated into a nucleic acid molecule that forms the product of association with the analyte. In the present invention, a single type of functionalized nucleotide, or a plurality of different types of functionalized nucleotides can be employed. Alternatively, or additionally, a functionalized nucleic acid or nucleic acid analog may already be present, which has been manufactured, for example by chemical or enzymatic synthesis, and which specifically binds, for example by hybridization, to the analyte to be detected.

In a preferred embodiment, the method comprises a primer extension reaction, optionally in combination with subsequent nucleic acid amplification steps, such as PCR. For example, at least one primer molecule can be provided that hybridizes under test conditions with a nucleic acid analyte to be detected, or its complement. The bound primer is then extended, in which a detectable extension product is obtained indicating the presence and / or amount of the nucleic acid analyte to be detected. According to this embodiment, functionalized primers and / or nucleotides or functionalized nucleotide analogs can be used for incorporation into the extension product.

Alternatively, and / or additionally, the method of the invention may comprise the use of functionalized hybridization probes, which hybridize under test conditions with the nucleic acid analyte to be detected, or its complement, in which the formation of a hybridization product indicates the presence and / or amount of the nucleic acid analyte to be detected.

The detection method of the invention can be carried out by any known nucleic acid detection protocols, for example involving the use of solid supports. For example, a solid support can be provided, for example a chip or matrix or a particulate material, such as a bead, to which a capture probe capable of hybridizing with the analyte to be detected is attached. The solid phase bound nucleic acid analyte can be detected using functionalized hybridization probes that hybridize with the nucleic acid analyte in a part of the sequence other than the capture probe does, and subsequently detect the bound hybridization probe. , for example with a metallization reagent. This method is particularly suitable for diagnostic applications in the agricultural and clinical field, for example for the detection of plant DNA and / or mRNA, for example 25 genetically modified plants, pathogen DNA or plant pests, etc.

In a specific embodiment, the detection may involve contacting the analyte association product and a functionalized compound comprising a photosensitizer group with a photosensitive medium, for example by transferring a sample or a sample aliquot in which a product may be present. of association on the photosensitive medium, for example by spotting, pipetting, etc. With irradiation, a transfer of energy is effected from the photosensitizer group to the photosensitive medium, so that marker groups such as metal cores, for example silver, are formed in the photosensitive medium in the presence, but not in the absence, of groups photosensitizers If necessary, the marker groups can be subjected to a development procedure, for example a chemical or photochemical development procedure according to photographic techniques. The photosensitive medium can be any solid support, or any supported material capable of forming marker groups, for example metal cores. Preferably, the photosensitive medium is a light sensitive medium, such as a light sensitive paper or a light sensitive emulsion or gel on a support material. More preferably, the photosensitive medium is a photographic medium, such as photographic paper. The radiation is carried out under conditions, for example wavelengths and / or intensity of the irradiation light, under which the formation of selective marker groups takes place in the presence of photosensitizer groups. Preferably, the irradiation takes place with infrared light and / or with longwave visible light 40, depending on the sensitivity of the medium. The irradiation wavelength may be, for example, 500 nm or greater, 520 nm or greater, 540 nm or greater, 560 nm or greater, 580 nm or greater for visible light, or 700 nm at 10 μm for infrared light

An important aspect of the invention is the detection of genetic variability, for example single nucleotide polymorphisms (SNPs). The genome of, for example, humans contains variations of the nucleotide sequence at an average frequency of up to 0.1%. Therefore, these variabilities provide excellent markers for the identification of genetic factors that contribute to the susceptibility of complex diseases [16, 17]. Although there is a wide range of techniques available for the detection of SNPs, most of these methods are slow, require large instrumentation, and amplification by PCR [18]. The present method, however, is both cheap and flexible enough to accommodate the screening needs of a low, moderate and high result with high speed, sensitivity and efficiency. There are several possibilities for selectively marking only the threads that are completely matched or the strands unpaired allowing the method of the invention to be used for the detection of SNPs.

For example, the detection of mismatches or nucleic acid mismatches, for example in SNPs, may comprise the use of functionalized hybridization probes that hybridize under the test conditions with the nucleic acid analyte to be detected or its complement, and it may comprise subjecting the hybridization product to a treatment method in which a hybridization product is dissolved that contains at least one mismatch, and in which the presence of an undissolved hybridization product indicates the presence and / or amount of a nucleic acid that has a completely complementary sequence (i.e. not unpaired) to the hybridization probe. 60

The treatment for the dissolution of the hybridization products containing mismatches may comprise a mismatch digestion treatment, that is, the use of enzymes that detect mismatches, which cleave the hybridization product depending on the presence of a mismatch. Suitable enzymes for such mismatch digestion treatment include mismatching glucosylases, such as those encoded by the hMSH2 and hMLH1 and Mut S, Mut L and Mut H genes. Additional proteins are MutY and 5 Mig.Mth1. Mig.Mth1 cuts T from a TG unpairing, MutY cuts A in an AG unpairing, and the TDG enzyme cuts T in a TG unpairing.

Alternatively, or additionally, hybridization products containing mismatches can be dissolved by a differential hybridization treatment that involves the adjustment of hybridization conditions, for example in view of temperature, saline concentration and / or washing with dimethylammonium chloride, in which a hybridization product 10 containing a mismatch is dissolved, and the completely complementary hybridization product remains stable.

In a still further embodiment, mismatches, for example SNPs, can be determined by selective enzyme-catalyzed primer elongation. To this end, a primer is provided, in which the 3 ′ end of the primer is located directly in the 5 ′ direction of a potential unpairing site in the mold analyte. An extension of the primer is only possible when a nucleotide is present which is complementary to the next base in the mold. By selecting a single type of functionalized nucleotide, and determining whether it is incorporated into the primer or not, the basis at the potential unpairing site can be determined.

The method of the invention comprises the detection of marker groups that are incorporated into an analyte association product with a functionalized compound. The marker groups are preferably selected from 20 metal deposition forming groups, for example aldehyde functionalized groups, from fluorescent or fluorescence forming groups, or from active redox groups.

The formation of metallic depositions requires the treatment of aldehyde groups with a metallization reagent, for example a reagent comprising atoms and / or metal ions selected from Ag, Au, Bi, Cu, Pd or Pt, which can be selectively deposited around aldehyde groups, for example by reduction. Preferably, the metallization reagent comprises an Ag + salt, such as an Ag-ammonium complex, that is the Tollens reagent. Other preferred examples of mealisation reagents are Cu (NO3) / I2, platinum complexes with terpyridine, such as [Pt (terpy) Cl] Cl, Pd (OAc) 2 or KAuCl4.

In addition, functionalized groups can also function as a handle to join other marker groups, such as fluorescent marker groups. For example, the marker compounds with amino groups can be coupled to aldehyde groups by reductive amination in the presence of a reducing agent such as sodium cyanoborohydride. Alternatively, the formation of hydrazones or oximes may be used to provide marker groups.

The detection of the marker groups can be carried out according to known methods. For example, metallic depositions can be determined qualitatively and / or quantitatively by optical methods and / or electrical methods. In a preferred embodiment, metal depositions on a solid surface can be determined by measuring electrical parameters, for example conductivity. The fluorescent marker groups can be determined qualitatively and / or quantitatively by known fluorescent measurement methods, for example excitation via a suitable light source such as a laser, and by detecting the emitted fluorescent light.

In a further embodiment, the invention comprises the detection of marker groups that are formed specifically at the site in a photosensitive medium in the presence of photosensitizing groups in a product of association of the analyte with a functionalized compound. The photosensitizer groups are preferably selected from fluorescent or luminescent groups. The photosensitizer groups can be incorporated directly into the association product or via a handle, for example via a Click reaction as explained in detail above. The photosensitive medium comprises groups that, when irradiated in the presence of photosensitizing groups, form detectable marker groups, such as metal nuclei, which can be developed according to standard photographic techniques, for example by chemical or photochemical development techniques.

The invention also relates to reagent kits for detecting analytes as described above, for example nucleic acid analytes, in a sample, comprising a functionalized compound having at least one functional group attached as described above.

The reagent kit may optionally contain an aldehyde-forming reagent or a second pair for a Click reaction, and a label or label-forming reagents or a photosensitive medium. The kits are preferably used in a method as indicated above.

In addition, the present invention relates to the use of a compound functionalized by Click of formula (II) as indicated above. The functionalized compounds may be building blocks for the chemical or enzymatic synthesis of nucleic acids or nucleic acid analogs or their precursors. Preferably, the compounds are nucleoside triphosphates, particularly ribose, 2′-deoxyribose or 2 ′, 3’-dideoxyribose triphosphates, or their analogs, which can be enzymatically incorporated into nucleic acids.

In addition, the compounds may be building blocks for the chemical synthesis of nucleic acids, or nucleic acid analogs such as peptide nucleic acids, morpholinic nucleic acids or blocked nucleic acids. For this purpose, phosphoramidites, H-phosphonates, nucleoside phosphorotrieters, or amino acids of nucleobases protected with Fmoc and / or Boc are suitable.

The invention also relates to a nucleic acid or nucleic acid analog molecule having at least 5 a compound of formula (II) incorporated therein: The molecule can be a nucleic acid selected from DNA and RNA, or an analogue of nucleic acid, for example selected from peptide nucleic acids, morpholinic nucleic acids or blocked nucleic acids, as described in detail above.

Furthermore, the invention relates to a method for synthesizing a nucleic acid or nucleic acid analog molecule, which comprises incorporating at least one nucleotide or nucleotide analog building block comprising a compound (II) in a nucleic acid molecule. or nucleic acid analog. The method may comprise a chemical synthesis and / or an enzymatic synthesis.

In addition, the invention relates to a metallization product of the nucleic acid or nucleic acid analog molecule as indicated above that is obtainable by treating a functionalized compound with nucleic acid-containing aldehyde with a metallization reagent as Indicate below in detail. fifteen

In addition, the invention relates to an association product of the nucleic acid or nucleic acid analog molecule as indicated above, with an analyte as described above. Preferably, the association product is a hybridization product with a complementary nucleic acid.

In a preferred embodiment, the methods and reagent kits of the present invention are used for agricultural applications. For example, the invention is suitable for the detection of plant nucleic acids, plant pathogens or plant pests such as viruses, bacteria, fungi or insects. In addition, the invention is suitable for detecting genetic variability, for example SNPs in plants or parts of plants, plant pathogens or plant pests, such as insects.

A further application is a detection or monitoring of resistance to herbicides, fungicides or pesticides, tolerances or intolerances, for example resistance, tolerances or intolerances in fungi, insects or plants in 25 organisms or populations of organisms. The invention is also suitable for rapid genotyping, for example for the rapid detection and / or differentiation of species or strains of fungi, insects, or plants. Furthermore, it is possible to detect and / or differentiate organisms or genetically modified strains, for example organisms or strains of fungi, insects or plants.

Due to the high sensitivity of the invention, an early diagnosis of pathogens is possible, that is, the diagnosis before the first symptoms of the presence of pathogens are visible. This is particularly important for the diagnosis of soybean rust (Phakospora pachyrizi) or other pathogens, for example Blumeria graminis, Septoria tritici or Oomycetes, or other pathogens for which control is only possible if their presence is detected before can be recognized visually.

Furthermore, the invention is suitable for medical, diagnostic and forensic applications, for example in human or veterinary medicine, for example for the detection of nucleic acids of pathogens, for example human pathogens or pathogens of cattle or companion animals.

Other preferred applications include the detection of genetic variability, for example SNPs, in humans, or the detection of resistance, tolerances or intolerances, or allergies to medications. In addition, the invention is suitable for genotyping, particularly the genotyping of humans, in order to determine mutations associated with increased predisposition or risk of disorders, allergies and intolerances. The invention can also be used for the detection of genetically modified organisms or strains, organisms or strains of bacteria or viruses, but also genetically modified farm animals, etc. The invention is particularly suitable for the rapid diagnosis of diseases, for example genetic diseases, allergic diseases, autoimmune diseases or infectious diseases. Four. Five

In addition, the invention is suitable for detecting the function and / or expression of genes, for example for research purposes.

Still a further embodiment is the use of the method for the protection of trademarks, for example to detect specific information encoded in products such as valuable goods such as plant protection products, drugs, cosmetics and fine chemicals (eg vitamins and amino acids) and products of beverage, 50 fuel products, for example gasoline and diesel, consumer electronics. In addition, you can mark the packaging of these and other products. The information is encoded by nucleic acids or nucleic acid analogs that have been incorporated into the product and / or the package of a product. The information may refer to the identity of the manufacturer, the production sites, the date of production and / or the distributor. By means of the present invention, rapid detection of product-specific data can be carried out. A sample 55 can be prepared from an aliquot of the product, which is then contacted with one or more sequence-specific functionalized hybridization probes, capable of detecting in the sample the presence of information encoded by the nucleic acid.

The invention is also suitable for the field of nutrients. For example, in the area of feed, animal nutrients, for example corn, are supplemented with a greater amount of preservatives, such as propionic acid. By applying the method of the invention, the addition of preservatives can be reduced. In addition, genomic analysis with the method of the invention allows the prediction of an individual's ability to use specific nutrients (nutrigenomic).

Still a further preferred embodiment refers to the field of epigenetics. This embodiment particularly relates to a DNA analysis, for example genomic DNA, with respect to the cytosine base methylation. In this embodiment, the DNA can be treated with a specific cytosine reagent, for example hydrazine and / or hydroxylamine. 10 Through treatment, a selective reaction of the cytosine or methylcytosine residues occurs. For example, treatment with hydroxylamine leads to a selective modification of cytosine residues. Preferably, the reagent is added in a sub-stoichiometric amount, in order to obtain a partial modification of, for example, cytosine residues. Subsequently, the treated DNA is analyzed, for example by a primer extension reaction, using at least one modified nucleic acid building block, as indicated above, for example a dU and / or 15 dC base. Preferably, a base modified by Click is used, for example a base modified by alkyne. The primer extension reaction gives a characteristic sequencing ladder that results from interruptions of the reaction in the modified dC or 5-methyl-dC bases (see Fig. 26).

A further preferred embodiment relates to the application of reporter nucleic acid molecules to a photosensitive medium, for example photographic paper or any other light sensitive medium. Preferably, the reporter molecules have a photosensitizer group and an extinguishing group. In the absence of analyte, the photosensitizer group is extinguished. For example, the reporter molecule may have a hairpin structure with the photosensitizer, and the extinguishing group at or near the terms of the molecule in close spatial relationship. When the reporter molecule is present as the hairpin structure, the photosensitizer group is extinguished (according to the known molecular beacon technique). In this way, a reporter molecule with an intact fork structure cannot sensitize when it radiates light to the photosensitive medium. In the presence of an analyte, the fork structure is broken. The analyte can be a strand of complementary nucleic acid, or an enzyme that cleaves the hairpin structure, or a protein that binds to the hairpin and thus breaks the structure. The photosensitizer group is separated from the extinguishing group, and thus is capable of photosensitization. In this case, the irradiation of light leads to a sensitization of the photographic medium, and thus to the detection of the analyte (see Fig. 27).

The invention is further explained by the following Figures and Examples.

Description of the Figures

Figure 1: Preferred binding sites of functional groups (R), for example aldehyde functionalized groups on different 7-dN-G, C, 7-dN-A and T nucleobases for enzymatic incorporation of marker groups into oligonucleotides.

Figure 2: Schematic representation of the synthesis of nucleoside building blocks functionalized with aldehyde, for solid phase synthesis of nucleic acids.

Figure 3a: Schematic representation of the synthesis of a nucleoside triphosphate functionalized with acetal (protected aldehyde), for the enzymatic synthesis of nucleic acids. 40

Figure 3b: An example of an alkyne functionalized nucleoside triphosphate for the effective chemistry of Click on DNA, in which n is preferably 0-4.

Figure 3c: An example of a functionalized triphosphate with protected aldehyde. The compounds shown in Figure 3 are suitable for efficient incorporation by PCR in DNA.

Figure 4: The results of a metallization reaction by contacting the Tollens reagent with an aldehyde modified base: (1) sugar solution as a reference, (2) DNA with another aldehyde modified group, (3) DNA with two aldehyde groups, and (4) unmodified DNA.

Figure 5: (1) Primer extension products stained with Ag (a), or using a standard fluorescent dye (b): I.1a: TTP modified with aldehyde at primer extension. I.1b: TTP modified with acetal at primer extension. 1.3a: unmodified DNA. Silver staining only positive on Line 1 and 2. I.1b-3b same gel but stained nonspecifically with a fluorescence dye. (II) A 2142 bp PCR product stained with a fluorescent dye (a) or with Ag (b): II2a: TTP modified with acetal in PCR. M: marker; II1b: TTP modified with aldehyde in PCR, 2: TTP modified with acetal in PCR, II3b: unmodified DNA.

Figure 6: Results of a dilution series with a synthetic DNA molecule that contains a single aldehyde functional group. Detection by staining with Ag. 55

Figure 7: Examples of nucleosides with different nucleobases possessing acetal (protected aldehyde) functions, which can be converted into corresponding phosphoramidites, H-phosphonates or tri-esters for chemical synthesis of nucleic acids, or 5'-triphosphates for enzymatic incorporation of nucleic acids.

Figure 8: Derivatization of aldehyde modified DNA or RNA with a fluorescence label, as an alternative to metallization via reductive amination. 5

Figure 9: Nucleoside and nucleotide monomers modified with alkyne (a, b), modified with azide (c) and modified with protected aldehyde (d, e), in which n is preferably 0-4.

Figure 10: Schematic representation of the synthesis of nucleotides and nucleotide triphosphates functionalized with alkynes.

Figure 11: Schematic representation of the synthesis of dendrimers functionalized with azide comprising 10 protected aldehyde groups.

Figure 12: Examples of azide and alkyne azide derivatives with marker groups or marker precursor groups for the Click reaction with nucleotides or nucleic acids modified with alkyne or azide.

Figure 13: At the top: MALDI-TOF analysis of the Click reaction with oligonucleotides containing modified base 2 (Figure 9b) and fluorescein azides as the Click pair (m: 495). At the bottom: gel electrophoresis of Click reactions of DNA modified with monomer 2 and fluorescein azide. A1: fluorescence image, Line 1: starting material before the Click reaction; 2, 3: crude reaction mixture; 4: azide under Click conditions; A2: same gel stained with SYBR green. B1: Line 1, 2: crude reaction mixture; 3: starting material; B2: same gel but stained with SYBR green.

Figure 14: Click reaction to introduce coumarin azide into DNA results in a fluorescent product 20 (right reaction tube).

Figure 15: An alkyne modified phosphoramidite (a), an alkyne modified nucleoside (b), and an azide modified galactose (c).

Figure 16: PCR assay using 200 µM of 8, dATP, dCTP and dGTP, 0.05 µg of genomic DNA from S. cerevisiae 499, 10x magnesium polymerase buffer, 0.3 mM of each of the primers and 1.25 U of 25 polymerases. Line 1) Vent, 2) Vent (exo-), 3) Pwo, 4) Taq (exo-) de Roche, 5) Therminator, 6) Taq de Promega and 7) marker.

Figure 17: PCR amplification of 2142 bp DNA with modified triphosphate 1 or 8 (Figure 10) replacing dTTP. Agarose gel (2%) was used and stained with ethidium bromide. A total of 35 PCR cycles were performed, using 45 seconds for each incubation. Line 1) NEB 2-log DNA ladder (0.1-10.0 kb), Line 2) negative control (minus 30 dTTP), Line 3) with substrate (1) replacing dTTP, 4) with substrate (8 ) replacing dTTP and 5) positive control (using dTTP).

Figure 18: HPLC chromatogram of (a) the enzymatic digestion of PCR fragments (318 mer) incorporating triphosphate 8 (Figure 10); (b) the enzymatic digestion of PCR fragments incorporating dTTP; (c) nucleoside 7 (Fig. 10). 35

Figure 19: Dilution series of 318 bp PCR fragments incorporating triphosphates 1 and 2 followed by the Click reaction "on gel" and silver staining. (a) Dilution series of PCR fragments incorporating 1 (Line 1 7.0 ng, Line 2 3.5 ng, Line 3 1.3 ng, Line 4 0.7 ng) and 2 (Line 5 7.0 ng, Line 6 3.5 ng, Line 7 1.3 ng, Line 8 0.7 ng) made on a PAE TBE-urea gel. The gel was subjected to a Click reaction "on the gel" with azide 3, and subsequent staining with silver. (b) Comparative detection of a dilution series of a PCR fragment incorporating 2 via 40 (i) the selective silver staining method (as (a)) and SYBR II green route (ii) (Line 1 7 ng, Line 2 3.5 ng, Line 3 1.3 ng, Line 4 0.9 ng, Line 5 0.5 ng, Line 6 0.3 ng). (c) Dilution series of PCR fragments incorporating 2 (Line 1 7 ng, Line 2 3.5 ng, Line 3 1.3 ng, Line 4 0.9 ng, Line 5 0.5 ng, Line 6 0 , 3 ng) and 2 (Line 1 7.0 ng, Line 2 3.5 ng, Line 3 1.3 ng, Line 4 0.7 ng) made on a PAE TBE-urea gel. The gel was then subjected to a Click reaction "on the gel" with aldehyde-modified azide 4, and subsequent staining with silver. (d) Dilution series of 45 PCR fragments incorporating 2 (Line 1 7 ng, Line 2 3.5 ng, Line 3 1.3 ng, Line 4 0.9 ng, Line 5 0.5 ng, Line 6 0.3 ng) performed on a PAE TBE-urea gel. The gel was then subjected to a Click reaction "on the gel" with aldehyde-modified azide 5, and subsequent staining with silver.

Figure 20: AFM photographs of natural DNA (a) and aldehyde modified DNA (b) after exposure to Ag staining conditions, and aldehyde modified DNA after staining with undeveloped Ag (c), after 2 minutes of 50 development (d) and after two times 2 minutes of development (e).

Figure 21: Schematic representation of a method of detection of an analyte, for example a DNA analyte, using the principles of black and white photography. Initial replication of the DNA analyte in the presence of a selective sequence primer produces a target sequence labeled with a sensitizer (S). After irradiation and subsequent photographic development, the analyte can be detected in a photosensitive medium, for example a photographic paper.

Figure 22: Representation of cyanine dyes (1), (2) and (3). Dilution series of cyanine dyes irradiated for 10 seconds using a projecting lamp from the top, equipped with a 520 nm cutting filter using (a) 1; (b) 2; and (c) 3, or irradiation for 5 minutes with an infrared lamp using (d) 1, (e) 2 and f (3). The 5 point experiments correspond to the following dilutions: Point 1: 1 x 10-11 moles, Point 2: 1 x 10-12 moles, Point 3: 3 x 10-13 moles, Point 4: 1 x 10-13 moles , Point 5: 3 x 10-14 moles, Point 6: 1 x 10-14 moles, Point 7: 3 x 10-15 moles, Point 8: 1 x 10-15 moles, Point 9: 3 x 10-16 moles , Point 10: 1 x 10-16 moles.

Figure 23: ODN-1 [5'-GCG ATT CGT TCG TCG C-3 '], ODN-2 [5'-CGC GAA TGA ACA GCG C-3' oligodeoxyribonucleotide point assays and ODN-3 [5 ' -GCG ACC GAT TCG C-3 '], all at 50 nmoles using (a) high energy irradiation and a cut filter (520 nm); and (b) low energy irradiation via prolonged exposure (5 minutes) using an infrared lamp.

Figure 24: Representation of cyanine dyes Cy5 and Cy5.5. Dilution series of cyanine dyes bound to the irradiated ODN for 10 seconds using a projecting lamp for the top, equipped with a 520 nm cutting filter using (a) ODN-4 [5'-Cy5-GCG CTG TTC ATT CGC G-3 ']; (b) ODN-5 [5’-Cy5.5-GCG CTG TTC 15 ATT CGC G-3 ’]; or irradiation for 5 minutes with an infrared lamp using (c) ODN-4; (d) ODN-5. The point experiments correspond to the following dilutions: Point 1: 1 x 10-11 moles, Point 2: 3 x 10-12 moles, Point 3: 1 x 10-12 moles, Point 4: 3 x 10-13 moles, Point 5: 1 x 10-13 moles, Point 6: 3 x 10-14 moles, Point 7: 1 x 10-14 moles, Point 8: 3 x 10-15 moles.

Figure 25: Schematic representation of the “Click and photography” methodology process. A sequence, for example a gene, of interest is specifically selected and amplified via PCR with alkyne modified triphosphates (dU * TP) and gene selective primers. These alkyne functions can be labeled via Click chemistry with an appropriate photosensitizer (S) that produces photosensitized DNA hybrids. Irradiation of the sample on photographic paper, and subsequent development, will provide a new method for ultrasensitive and gene-specific detection. 25

Figure 26: Schematic representation of a method to identify DNA methylation patterns by specific chemical modifications of cytosines. A DNA analyte is reacted with a specific cytosine (or methylcytosine) modifying agent, for example hydroxylamine, preferably in sub-stoichiometric amounts, in order to give partially modified cytosine residues (C *). A primer extension reaction with an alkynyl modified base is carried out. This reaction ends in the modified cytosine residues, resulting in a "ladder" of extension products having a characteristic length. After separating the extension products from the primer, for example on a gel, a Click reaction is carried out with an azide modified marker group, for example a modified sugar, and a subsequent detection of the marker group (for example via staining with silver). The methylation pattern of the DNA sample can be determined by comparing the extension products of a methylated DNA sample with the extension products of a non-methylated DNA sample, which has undergone the same treatment.

Figure 27: Schematic representation of a molecular beacon detection method in a photosensitive medium. An informant molecule is provided having a fluorescence group (F) and an extinction group (Q). In the absence of analyte, the reporter molecule has a hairpin structure, and the fluorescence is extinguished. In the presence of the analyte, the extinguishing group is removed from the fluorescence group, which is no longer extinguished (F *). The sample is irradiated in the presence of a photographic medium, such as photographic paper or other light sensitive material, and is developed.

Examples

Example 1 (Reference)

Synthesis of nucleosides and nucleotides modified with aldehyde

Nucleosides and nucleoside triphosphates were synthesized with nucleobases modified with aldehyde from appropriately modified nucleosides having a leaving group such as I, preferably in position 5 of the pyrimidine bases, for example T, U and C, or in the position 7 of purine bases, for example 7-desaza G, 7-desaza A, G and A, as shown in Figure 1. All modifications in these positions protrude from the main DNA slot.

The synthesis of an aldehyde-modified T (U) nucleoside phosphoramidite, first via 50-alkylated Sonogashira-alkylation [20], followed by reaction with the appropriate functional groups, was carried out as shown in Figure 2. The function Aldehyde remained protected as an acetal group. The acetal protecting group can be removed by treatment with acid. The modified nucleoside phosphoramidite can be incorporated into nucleic acids by chemical synthesis.

The synthesis of an acetal modified nucleoside triphosphate was carried out as shown in Figure 3a. Figure 3b shows other nucleoside triphosphates modified with alkyne (for the Click reaction), with acetal and with hemiacetal. Modified nucleoside triphosphates can be incorporated into nucleic acids by enzymatic synthesis.

The use of Pd-based C-C formation reactions, such as by Sonogashira / Suzuki [20], provides a general synthetic approach to the synthesis of nucleosides and nucleotides modified with aldehyde with different aldehyde groups with different modified nucleobases. In addition, aldehyde groups with different steric, electronic and functional characteristics can be synthesized.

Example 2 (Reference)

Incorporation of nucleosides and aldehyde modified nucleotides into oligonucleotides 10

2.1 Incorporation through chemical synthesis

The incorporation of nucleoside phosphoramidite modified with acetal / aldehyde was achieved by conventional solid phase synthesis [21]. Several DNA oligonucleotides containing one or multiple acetal / aldehyde modified nucleobases were prepared, as depicted in Table 1. Deprotection of the acetal groups gave the aldehyde modified DNA oligonucleotide. fifteen

The oligonucleotides were tested for their ability to form base pairs with complementary strands of DNA, in order to obtain double stranded DNA molecules. It was found that the reduction in duplex stability due to the presence of the aldehyde group is low. The melting point of the double chains is reduced by about 2 ° C per modification. This amount of destabilization is, however, completely acceptable for the intended applications. twenty

Table 1: Oligodeoxynucleotides synthesized by solid phase synthesis

 Tm [ºC]

 one

 TTTTTTXTTTTT

 2

 TTTTTTXXTTTTT

 3

 GCCGAXGCGC 53.8 (56.5 control not modified)

 4

 GCGXATAXATAXTCGC 48.4 (53.7 unmodified control)

 5

 TTXTTXTTXTTXTTX

2.2 Incorporation by enzymatic methods

Acetal modified triphosphate monomers were prepared according to Figure 3. The most selective method of synthesizing triphosphates is the procedure followed by Ludwig and Eckstein [11, 12]. This procedure is slightly acidic, so a mixture of aldehyde- and acetal-TTP is obtained, which can be separated by HPLC if desired [13].

Studies were performed with both acetal and aldehyde modified triphosphates and with a variety of different DNA polymerases. They showed that all polymerases accept modified triphosphate as a substrate, and selectively incorporate the modified base. It was also seen that the multiple additions were not problematic. The following polymerases were tested in primer and PCR extension studies. Experiments were carried out with thymidine protected with acetal, and also with thymidine functionalized with unprotected aldehyde.

 Primer extension

 PCR

 Therminator (NEB)

 Pfx (A)

 Vent

 Taq (A)

 Vent (exo-)

 Vent (B)

 Taq

PCR studies were also carried out with TPP substituted by synthetic triphosphates (acetal and aldehyde). The PCR experiment showed that the modified base can be incorporated into a complete gene of 2400 base pairs in length, stating that genes specifically labeled with hundreds of nucleation sites derived from aldehyde can be created.

Example 3 (Reference)

Detection of oligonucleotides and labeled genes

A preferred method for metal deposition on aldehydes is based on the Tollens test [14] 5

R-CHO + 2 Ag [NH3] OH → R-COO-NH4 + + 2 Ag (s) + H2O + NH3

In this reaction, a silver-ammonium complex is reduced by the aldehyde. If the concentration of the aldehyde is high, a solid silver film can be seen inside the reaction tube.

Both the nucleoside containing the aldehyde and the chemically synthesized aldehyde-modified nucleic acids could be detected in a silver staining test indicating aldehyde functions (Fig. 4). A strand 10 of unlabeled DNA on the other hand gave a clearly negative result under the same conditions, showing that the aldehyde selectively introduced into specific DNA sequences allows the selective detection of these modified sequences.

The detection was also carried out on gels by staining with aldehyde modified nucleic acids with silver. The aldehyde groups in the DNA were sufficient to reduce the silver ions in a gel. The seeds formed by the process on the DNA could be developed until a clear black band formed by the deposited silver could be observed in the gel.

This was demonstrated by staining aldehyde modified nucleic acids from primer extension reactions and from PCR (Figure 5 (I) and (II), respectively).

As shown in Fig. 5, the DNA containing the modified nucleoside is stained exclusively with Ag, 20 while the unmodified DNA remains unstained. This is true for the extended primer product and for the PCR product. The staining procedure is based on the SilverXPress kit (Invitrogen), but with a more concentrated Tollens solution. Staining is also possible after other procedures.

Example 4 (Reference)

Sensitivity determination 25

In order to determine the sensitivity of the detection method, dilution experiments were carried out (Figure 6). It was found that staining of DNA functionalized with aldehyde with a standard Ag staining kit was at least five times more sensitive than staining with ethidium bromide (,50.5 ng of DNA with a length of 50 bp).

This tremendously impressive sensitivity is obtained even with normal visual detection.

Example 5 30

Synthesis of nucleosides and nucleotides modified with azide and with alkyne

Nucleosides and nucleoside triphosphate with bases modified with azide and alkyne were substantially synthesized as described in Example 1.

5.1 Synthesis of alkyne modified nucleotides

A detailed reaction scheme for the synthesis of nucleoside triphosphate modified with alkyne is shown in Figure 10.

The preparation of nucleoside (1) and its corresponding triphosphate (2) was carried out as previously [23, 24].

At a conscious degassed solution of 3 (1.00 g, 1.72 mmol) [3], Pd (PPh3) 4 (0.198 g, 0.172 mmol) and CuI (0.065 g, 0.344 mmol) in DMF (3 ml) Degassed N, N-diisopropylethylamine (1.5 ml, 8.59 mmol) was added, and the reaction mixture was stirred at room temperature for 10 minutes. To the reaction mixture was added dropwise a degassed solution of 4-pentin-1-ol (320 µl, 3.44 mmol) in DMF (1 mL), for 1 hour. After finishing the addition, the reaction mixture was stirred at room temperature overnight. After concentrating in vacuo, the crude mixture was diluted with ethyl acetate (200 ml), and the organic layer was washed with brine (3 x 50 ml) followed by water (50 ml). The organic layer was dried (MgSO4), filtered, and concentrated in vacuo. Flash column chromatography (SiO2) eluting with a gradient of ethyl acetate: isohexane (1: 1), followed by ethyl acetate: isohexane (2: 1), provided 4 (0.790 g, 86%) as a foam pale yellow 1H NMR (CDCl3, 300 MHz): δ 0.01 (s, 3 H, OSiCH3), 0.00 (s, 3 H, OSiCH3), 0.05 (s, 3 H, OSiCH3), 0.07 ( s, 3 H, OSiCH3), 0.81 (s, 6 H, OSi (CH3) 3), 0.85 (s, 6 H, OSi (CH3) 3), 1.74 (m, 2 H, CH2 -CH2CH2-OH), 1.95 (m, 1 H, H-2β), 2.20 (m, 1 H, H-2α), 2.42 (t, 2H, J = 6.0 Hz, C ≡C-CH2CH2-), 3.67 (dd, 1 H, J = 11.4, 2.1 (CDCl3, 75.5 MHz): δ 5.4 (SiCH3), -5.3 (SiCH3), -4.8 (SiCH3), -4.6 (SiCH3), 16.7 (C≡C-CH2CH2), 18.0 (SiC (CH3) 3), 18.5 (SiC (CH3) 3), 26 , 1 (SiC (CH3) 3), 26.4 (SiC (CH3) 3), 31.5 (CH2-CH2-CH2-OH), 42.3 (C-2 '), 61.9 (CH2- CH2CH2-OH), 63.3 (C-5 '), 72.4 (C≡C), 72.7 (C-3'), 86.0 (C-1 '), 88.7 (C- 4 '), 95.0 (C≡C), 100.9 (C-5), 101.0 (C≡C), 141.9 (C-6), 149.6 (C-2), 162 , 4 (C-4) HRMS (ESI, + ve) calcd for C26H46N2NOO6Si2 561.2792 [M + Na] +, found 561.2803.

To a stirred solution of 4 (0.300 g, 0.56 mmol) in dichloromethane (5 ml) was added a solution of Dess-Martin 5-periodine (0.378 g, 0.89 mmol) in dichloromethane (5 ml), drop a drop for 5 minutes at room temperature under nitrogen atmosphere. The reaction mixture was stirred for 2 hours, followed by paralysis with saturated sodium thiosulfate solution (10 ml). The crude mixture was then diluted with dichloromethane (200 ml), and the organic layer was washed with brine (2 x 30 ml) and water (2 x 30 ml). The organic layer was then dried (MgSO4), filtered and concentrated in vacuo. Column chromatography (flash silica), eluting with 35% ethyl acetate in 10 isohexane, provided 5 (0.193 g, 65%) as a pale yellow foam. 1H NMR (CDCl3, 600 MHz): δ 0.07 (s, 3 H, OSiCH3), 0.00 (s, 3 H, OSiCH3), 0.05 (s, 3 H, OSiCH3), 0.06 ( s, 3 H, OSiCH3), 0.81 (s, 6 H, OSi (CH3) 3), 0.85 (s, 6 H, OSi (CH3) 3), 1.95 (m, 1H, H- 2β), 2.21 (m, 1H, H-2α), 2.61 (t, 2 H, J = 7.2 Hz, -CH2CH2-), 2.67 (t, 2H, J = 7.2 Hz, -CH2CH2-), 3.68 (dd, 1 H, J = 11.4, 2.4 Hz, H-5 '), 3.81 (dd, 1 H, J = 11.4, 2, 4 Hz, H-5 '), 3.89 (m, 1 H, H-4'), 4.32 (m, 1 H, H-3 '), 6.20 (dd, 1 H, J = 7.2, 6 Hz, H-1 '), 7.84 (s, 1 H, H-6), 8.99 (bs, 1 H, NH), 9.72 (s, 1 H, CHO) . 13C NMR (CDCl3, 150.8 15 MHz): δ 3.8 (SiCH3), -3.7 (SiCH3), -3.1 (SiCH3), -2.9 (SiCH3), 14.4 (C≡ C-CH2CH2), 19.7 (SiC (CH3) 3), 20.1 (SiC (CH3) 3), 27.4 (SiC (CH3) 3), 27.7 (SiC (CH3) 3), 43 , 7 (C-2 '), 44.1 (CH2-CH2-CHO), 64.6 (C-5'), 74.0 (C-3 'and C≡C), 87.5 (C- 1 '), 90.0 (C-4'), 94.3 (C≡C), 101.9 (C-5), 143.8 (C-6), 150.9 (C-2), 163.7 (C-4), 201.6 (CHO). HRMS (ESI, + ve) calc. for C26H44N2NaO6Si2 559.2635 [M + Na] + found 559.2634.

To a solution of 5 (0.530 g, 1.00 mmol) and K2CO3 (0.269 g, 1.98 mmol) in dry methanol (20 ml) was added a solution of dimethyl ester of 1-diazo-2-oxo- acid propyl phosphonic (0.261 g, 1.19 mmol) in dry methanol (5 ml) at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight, filtered and concentrated in vacuo. The crude mixture was dissolved in ethyl acetate (200 ml), and the organic layer was washed with brine (2 x 30 ml) and water (2 x 30 ml), dried (MgSO4) and concentrated in vacuo. Column chromatography (flash silica), eluting with 20% ethyl acetate in isohexane, provided 6 (0.290 g, 55%) as a colorless foam. 1H NMR (CDCl3, 600 MHz): δ 0.00 (s, 3 H, OSiCH3), 0.01 (s, 3 H, OSiCH3), 0.06 (s, 3 H, OSiCH3), 0.07 ( s, 3 H, OSiCH3), 0.82 (s, 6 H, OSi (CH3) 3), 0.86 (s, 6H, OSi (CH3) 3) 1.93 (m, 1H, H-2β) , 1.95 (s, 1 H, C≡CH, 2.22 (m, 1H, H-2α), 2.39 (dt, 2H, J = 8.4, 2.4 Hz, -CH2CH2-) , 2.55 (t, 2 H, J = 8.4 Hz, -CH2CH2-), 3.68 (dd, 1 H, J = 11.4, 2.4 Hz, H-5 '), 3, 81 (d, 1 H, J = 11.4, 2.4 Hz, H-5 '), 3.89 (m, 1 H, H-4'), 4.33 (m, 1 H, H- 3 '), 6.20 (dd, 1 H, J = 7.8, 6 Hz, H-1'), 7.85 (s, 1 H, H-6), 8.33 (bs, 1 H , 13C NMR (CDCl3, 150.8 MHz): δ 7.0 (SiCH3), -6.9 (SiCH3), -6.4 (SiCH3), -6.2 (SiCH3), 16.5 ( C≡C-30 CH2CH2), 16.9 (C = C-CH2CH2), 17.0 (SiC (CH3) 3), 18.2 (SiC (CH3) 3), 24.2 (SiC (CH3) 3 ), 24.5 (SiC (CH3) 3), 40.4 (C-2 '), 61.4 (C-5'), 67.9 (C≡CH), 70.8 (C-3 ' ), 70.9 (C≡C), 80.9 (C = C), 84.1 (C-1 '), 86.8 (C-4'), 91.2 (C≡C), 98 , 7 (C-5), 140.6 (C-6), 147.5 (C-2), 159.9 (C-4) HRMS (FAB, + ve) calcd for C27H43N2O6Si2 + H 533, 28670 [M + H] + found 533.26009.

To a cooled solution (0 ° C) of 6 (0.270, 0.51 mmol) in THF (5 ml) was added TBAF (1.0 M, 1.52 ml, 1.52 mmol) under a nitrogen atmosphere. The reaction mixture was stirred for 3 hours, paralyzed with glacial acetic acid (1.0 ml) and concentrated in vacuo. Column chromatography (flash silica), eluting with 10% methanol in ethyl acetate, provided 7 (0.127 g, 82%) as a colorless oil. 1H NMR (d6-DMSO, 400 MHz): δ 1.88 (s, 1 H, C≡CH), 2.07 (m, 2 H, H-2 '), 2.36 (dt, 2 H, J = 7.2, 2.4 Hz, -CH2CH2-), 2.54 (t, 2 H, J = 6.8 Hz, -CH2CH2-), 3.53 (dd, 1 H, J = 12, 0, 4.0 Hz, H-5 '), 3.58 (dd, 1 H, J = 12.0, 4.0 Hz, H-5'), 3.76 (m, 1 H, H- 4 '), 4.19 (m, 1 H, H-3'), 5.07 (bs, 1 H, OH), 5.23 (bs, 1 H, OH), 6.08 (t, 1 H, J = 6.4 Hz, H-1 '), 8.11 (s, 1 H, H-6), 11.55 (bs, 1 H, NH). 13C NMR (CDCl3, 150.8 MHz): δ 17.6 40 (C≡C-CH2CH2), 18.9 (C≡C-CH2CH2), 39.5 (C-2 '), 60.7 (C -5 '), 70.0 (C-3'), 73.7 (C≡CH), 75.1 (C≡C), 84.6 (C-1 '), 87.3 (C-4 '), 89.1 (C = C), 93.0 (C≡C), 100.2 (C-5), 144.7 (C-6), 151.0 (C-2), 163, 2 (C-4). HRMS (FAB, + ve) calc. for C15H16N2O5 + Na [M + Na] + 327.28771 found 327.09635. HRMS (FT-ICR MS -) calc. for C15H16N2O5 + AcO- [M + AcO -] - 363,1198 found 363,1221, MS-MS calc. for C15H15N2O5- [M-] 303.1 found 303.1.

To a cooled solution (0 ° C) of 7 (0.060 g, 0.197 mmol) and 1,8-bis (dimethylamino) naphthalene (proton sponge, 0.068 45 g, 0.316 mmol) trimethyl phosphate (2 ml) phosphorus oxychloride was added (22 µl, 0.237 mmol) drop by drop for 5 min. in an atmosphere of nitrogen. The reaction mixture was stirred for 3 hours at 0 ° C. To the reaction mixture was added a solution of tributylammonium pyrophosphate (0.080 g, 0.237 mmol) in dry DMF (2.0 ml), and stirred for 1 min., Followed by paralysis with triethylammonium bicarbonate (1.0 M , 20 ml, pH = 8.5). The reaction mixture was stirred for 2 h and freeze dried overnight. RP-HPLC purification (0-50% 0.1 M triethylammonium acetate → 20:80 50 H2O: MeCN with 0.1 M triethylammonium acetate gradient for 45 min at a flow rate of 5 ml.min.- 1) produced 8 (17.7 min.) As the triethylammonium salt. 31P NMR (D2O, 81 MHz): δ 22.4 (t, 1 P, J = 19.8 Hz, P-β), -10.5 (d, 1 P, J = 20.3 Hz, P- α), -9.7 (d, 1 P, J = 19.8 Hz, Py). MALDI-TOF (ATT matrix, negative mode): 271 [M + 2 H] 2-, 542 [M + 2 H] -.

5.2 Synthesis of Click reaction pairs functionalized with dendrimeric azide

A detailed reaction scheme for the synthesis of dendrimers functionalized with azide containing a plurality of hemiacetal groups (protected aldehyde groups) is shown in Figure 11.

General procedure for the triazole ligation reaction catalyzed by Cu (I)

To a solution of 9 (7.31 g, 0.0256 mol), 10 (3.00 g, 0.0128 mol) in THF: H2O (3: 1, 40 ml) was added a solution of CuSO4 (0.103 g , 0.641 mmol) in H2O (3 ml), followed by solid sodium ascorbate (0.253 g, 1.28 mmol). The reaction mixture was stirred overnight at room temperature. The suspension was diluted with H2O (50 ml), cooled to 0 ° C and treated with concentrated NH4OH (5 ml) for 10 minutes. The reaction mixture was diluted with dichloromethane (500 ml), and the organic layer was washed with brine (2 x 50 ml), followed by H2O (2 x 50 ml). The organic layer was retained, dried (MgSO4) and concentrated in vacuo. Column chromatography (ultrafast silica), eluting with 1: 1 ethyl acetate: petroleum ether, provided 9 (2.76 g), mono-Click product (11, 3.94 g) as a colorless foam, followed by 12 (4.92 g, 48%). The mono-Click product (11) was then reacted with an equivalent of 9 to provide 12 quantitatively after treatment and without further purification required. 10

1H NMR (CDCl3, 600 MHz): δ 1.29 (s, 6 H, C-CH3), 1.35 (s, 6H, C-CH3), 1.38 (s, 6 H, C-CH3) , 1.49 (s, 6 H, C-CH3), 4.19 (m, 4H, H4 '/ H5'), 4.32 (dd, 2 H, J = 4.8, 2.4 Hz, H 2 '), 4.46 (dd, 2H, J = 14.4, 8.4 Hz, H6 ”), 4.50 (s, 2H, -CH2-), 4.62 (m, 2 H, H 3 '), 4.64 (m, 2 H, H 6'), 5.18 (s, 4H, -CH2-), 5.51 (d, 2 H, J = 4.8 Hz, H 1 '), 6.59 (t, 1 H, J = 1.8 Hz, Ar-H), 6.64 (d, 2H, J = 1.8 Hz, Ar-H), 7.79 (s, 2H, C = CH-). 13C NMR (CDCl3, 150.8 MHz): δ 24.4 (C-CH3), 24.7 (C-CH3), 25.9 (C-CH3), 26.0 (C-CH3), 46, 1 (CH2-), 50.6 (-CH6 '/ H6 ”), 62.2 (Ar-CH2-), 67.0 (CH4'), 70.1 (CH2 '), 70.5 (CH3' ), 70.9 (CH5 '), 96.0 15 (CH1'), 102.0 (Ar-CH), 107.9, 109.1, 110.0, 124.0 (C = CH), 139 , 6, 143.4, 160.0. ESI-MS m / z 806 [M + H] +.

The reaction of 13 (1,539, 1.88 mmol) and 10 (0.220 g, 0.94 mmol) in the presence of CuSO4 (0.008 g, 0.047 mmol) and sodium ascorbate (0.019 g, 0.094 mmol) provided 14 (1.65 g, 95%) as a colorless foam after column chromatography (ultrafast SiO2; ethyl acetate: methanol 9: 1). 1H NMR (CDCl3, 600 MHz): δ 1.26 (s, 12H, C-CH3), 1.27 (s, 6 H, C-CH3), 1.34 (s, 12 H, C-CH3) , 1.35 (s, 12 H, C-CH3), 1.48 (s, 6 H, C-CH3), 4.19 (m, 8 H, H4 '/ H5'), 20 4.32 ( m, 4 H, H2 '), 4.46 (m, 4H, H6 ”), 4.47 (s, 2 H, -CH2-), 4.62 (m, 4 H, H 3'), 4 , 64 (m, 4H, H 6 '), 5.12 (s, 8 H, -CH2-), 5.15 (s, 4 H, -CH2-), 5.40 (s, 4 H, - CH2-), 5.49 (bd, 4 H, J = 4.8 Hz, H 1 '), 6.50 (m, 4 H, Ar-H), 6.55 (m, 2 H, Ar- H), 6.58 (m, 1 H, Ar-H), 6.61 (m, 2 H, Ar-H), 7.59 (s, 2 H, C = CH-), 7.78 ( s, 4H, C = CH-). 13C NMR (CDCl3, 150.8 MHz): δ 24.4 (C-CH3), 24.9 (C-CH3), 25.9 (C-CH3), 26.0 (C-CH3), 45, 9 (CH2-), 50.4 (-CH6 / H6 ”), 53.9 (Ar-CH2-), 61.8 (Ar-CH2-), 61.9 (Ar-CH2-), 67.0 (CH4 '), 70.1 (CH2'), 70.5 (CH3 '), 71.1 (CH5'), 96.1 (CH1 '), 101.6, 101.7, 107.2, 108 , 0, 109.0, 109.9, 122.9, 25 124.2, 136.7, 140.0, 143.2, 144.1, 159.5, 160.0. MALDI-TOF (ATT, positive mode): m / z 1859 [M + H] +.

General procedure for the conversion of dendritic chlorides into azides

To a solution of 12 (4.84 g, 6.01 mmol) in acetone: water (4: 1, 100 ml) NaN3 (0.586 g, 9.01 mmol) was added and heated to reflux under a nitrogen atmosphere during 3 hours. The reaction mixture was diluted with ethyl acetate (600 ml), and the organic layer was washed with brine (2 x 50 ml) followed by water (2 x 50 ml). The organic layer was retained, dried (MgSO4) and concentrated in vacuo to yield 13 (4.72 g, 97%) as a white amorphous solid.

1H NMR (d6-acetone, 400 MHz): δ 1.28 (s, 6H, C-CH3), 1.35 (s, 6 H, C-CH3), 1.36 (s, 6 H, C- CH3), 1.44 (s, 6 H, C-CH3), 4.30 (dd, 2 H, J = 3.6, 2.0 Hz, H 6 '/ 6 ”), 4.32 (dd , 2 H, J = 3.2, 1.6 Hz, H 5 '), 4.36 (dd, 2 H, J = 7.6, 1.6 Hz, H 4'), 4.39 (dd , 2 H, J = 4.8, 2.4 Hz, H 2 '), 4.61 (d, 2 H, J = 3.6 Hz, H 6' / 6 ”), 4.64 (s, 2H, -CH2), 4.70 (dd, 2 H, J = 7.6, 2.4 Hz, H 3 '), 5.20 (s, 4 H, -CH2-), 5.47 (d , 2 H, J = 4.8 Hz, H 1 '), 6.73 (m, 3 H, Ar-H), 8.07 (s, 2 H, C = CH). 13C NMR (d6-acetone, 150.8 35 MHz): δ 25.7 (C-CH3), 26.1 (C-CH3), 27.2 (C-CH3), 27.4 (C-CH3) , 47.6 (CH2-), 52.0 (-CH6 '/ H6 ”), 63.4 (Ar-CH2-), 68.8 (CH4'), 72.0 (CH2 '), 72.5 (CH3 '), 72.9 (CH5'), 98.2 (CH1 '), 103.3 (Ar-CH), 109.9 (Ar-CH), 110.4, 111.2, 126.3 (C = CH), 141.9, 144.8, 161.8. HRMS (ESI, + ve) calc. for C37H49N9O12Na 834.3398 [M + Na] + found 834.3404.

The reaction of 14 (1.51 g, 0.81 mmol) and NaN3 (0.079 g, 1.22 mmol) provided 15 (1.45 g, 96%) as a white amorphous solid. 1H NMR (CDCl3, 600 MHz): δ 1.27 (s, 12 H, C-CH3), 1.34 (s, 12 H, C-CH3), 1.37 (s, 12 H, C-CH3 ), 1.48 40 (s, 12 H, C-CH3), 4.19 (m, 8 H, H4 '/ H5'), 4.24 (s, 2 H, -CH2-), 4.32 (m, 4 H, H2 '), 4.46 (dd, 4 H, J = 13.8, 8.4 Hz, H6 ”), 4.62 (m, 4 H, H3'), 4.64 (m, 4 H, H 6 '), 5.12 (s, 4H, -CH2-), 5.18 (s, 8 H, -CH2-), 5.43 (s, 4 H, -CH2- ), 5.49 (bd, 4 H, J = 4.8 Hz, H 1 '), 6.50 (m, 2 H, Ar-H), 6.55 (m, 4 H, Ar-H) , 6.58 (m, 1 H, Ar-H), 6.60 (m, 2 H, Ar-H), 7.78 (s, 2 H, C = CH-), 7.79 (s, 4 H, C = CH-). 13C NMR (CDCl3, 150.8 MHz): δ 24.4 (C-CH3), 24.8 (C-CH3), 25.8 (C-CH3), 25.9 (C-CH3), 45, 9 (CH2-), 50.4 (-CH6 '/ H6 ”), 54.6 (Ar-CH2-), 62.0 (Ar-CH2-), 62.1 (Ar-CH2-), 67, 1 (CH4 '), 70.3 (CH2'), 70.7 (CH3 '), 71.1 (CH5'), 96.2 45 (CH1 '), 101.7, 101.8, 107.4 , 107.7, 109.0, 109.9, 124.0, 124.2, 137.8, 143.1, 143.4, 144.1, 159.7, 159.9. HRMS (ESI, + ve) calc. for C87H110N21O26Na 1886.7829 [M + Na + H] +, found 1887.7862.

General procedure for deprotection of dendritic isopropylidene

To a mixture of TFA: water (1: 1; 10 ml) was added 13 (0.020 g, 24.7 µmoles) under a nitrogen atmosphere. The reaction mixture was heated to 50 ° C for 4 hours, followed by concentration in vacuo. Water (20 ml) was added to the mixture, and the aqueous layer was washed several times with DCM (3 x 20 ml), retained and lyophilized overnight to produce 16 (0.015 g, 63%) as a foam pale yellow MALDI-TOF (ATT, positive mode): m / z 652 [M + H] +.

Reaction of 15 (1.40 g, 0.75 mmol) with TFA: water (1: 1; 20 ml) provided 17 (1.05 g, 90%) as a pale yellow amorphous solid. MALDI-TOF (ATT, positive mode): m / z 1545 [M + H] +.

  55

Example 6

Incorporation of nucleosides and nucleotides modified with azide and with alkyne in nucleic acids

Azide and alkyne modified nucleosides and nucleotides are effectively incorporated into nucleic acids by chemical or enzymatic synthesis substantially as described in Example 2. The resulting modified nucleic acids can be specifically labeled according to the desired application, for example by fluorescent labeling 5 or metal deposition

This labeling strategy is a fast and extremely sensitive method for the detection of nucleic acids, nucleic acid sequences and genes, even without the need for PCR amplification.

In a first experiment, to investigate the behavior of the Click reaction in a DNA architecture, nucleosides 1 and 2 (Figure 9a and b) were prepared, and incorporated into oligonucleotides via standard solid phase 10 synthetic protocols. The effectiveness of the Click reaction was investigated using benzyl azide, CuSO4, or reducing agent (ascorbate or TCEP) and a ligand that stabilizes copper (I). The results of these are summarized in Table 2. Oligonucleotides comprising rigid base 1 consistently gave lower yields of the Click product compared to flexible alkyne 2. The temperature (10 to 40 ° C) did not affect the yields of the reactions. Click. fifteen

The effectiveness of the Click reaction was then evaluated as a function of the type of azide showing properties suitable for detection (Figure 12). Fluorescein azides are extremely sensitive fluorescent markers (almost unit fluorescent quantum yield), while coumarin azide provides a Click marker test as fluorescence is triggered with triazole formation [19].

Table 2: Summary of Click reactions using benzyl azide, CuSO4 and TCEP. twenty

 ODN sequence

 X = 1 X = 2

 Performance (%)

 Starting material

 Desired product Other products Starting material Desired product Other products

 3’-GCG CTT ACX TGT CGC G-5 ’

 3 97 - - 100 Azide reduction

 3’-GCG CTT ACX XGT CGC G-5 ’

    84 16 (monoclick) - 100 Azide Reduction

 3’-GCG CTT ACX TGX CGC G-5 ’

    94 6 (monoclick) - 100 Azide Reduction

 3’-GCG CTX ACX TGX CGC G-5 ’

    84 16 (bisclick) NA NA NA

Consistent with the experiments involving the use of benzyl azide, oligodeoxynucleotides comprising rigid alkyne gave lower Click conversion yields. Oligonucleotides containing the flexible alkyne provide, on the contrary, an almost quantitative conversion according to HPLC and MALDI-TOF. DNA labeling was again possible directly on the gel. The DNA containing the modified nucleobase was separated from another 25 strands of DNA by gel electrophoresis, and the gel was then treated with fluorescein azide and Cu (I) to carry out the Click reaction. Washing the gel removed the excess fluorescein, leaving the stained DNA in the gel (Figure 13).

From these experiments it can be clearly deduced that the DNA containing the alkylated modified bases can be selectively labeled with fluorescein. The green CYBR colors the DNA in a non-specific way, and therefore also marks the unmodified DNA before the Click reaction, as in line 1 of A2 and line 3 of B2 (Figure 13).

Fluorescein is a molecule that fluoresces strongly both as a monomer and after a Click reaction with DNA. After the Click reaction with fluorescein, the gel must therefore be washed in order to remove excess fluorescein, which leaves the DNA stained.

Coumarin is an alternative fluorescent molecule, which however does not fluoresce as a monomer. It begins to emit fluorescence only after the Click reaction, when the azide becomes triazole. The fluorescence of coumarin is effectively turned on after the Click reaction, as seen in the experiment below in reaction tubes (Figure 14). A similar experiment can be performed on the gel.

In an additional experiment, the effectiveness of the Click reaction with oligodeoxynucleotides (ODN) incorporating nucleoside 2 was investigated (Figure 15). This nucleoside was incorporated into oligonucleotides via standard solid phase synthetic protocols, using the corresponding phosphoramidite 1 (Figure 15). Table 3 shows the 10 oligonucleotides comprising a single alkyne reporter group or multiple alkyne reporter groups.

Table 3

 ODN sequences

 X = 19

 Mass

 Starting material

 Product

 3’-GCG CTT ACXTGT CGC G-5 ’

 ODN1 4952 (calc.) 4955 (obt.) ODN1-P 5157 (calc.) 5157 (obt.)

 3’-GCG C-7 ACX XGT CGC G-5 ’

 ODN2 5042 (calc.) 5042 (obt.) ODN2-P 5452 (calc.) 5455 (obt.)

 3’-GCG CTT XXX XXX CGC G-5 ’

 ODN6 5407 (calc.) 5413 (obt.) ODN6-P 6637 (calc.) 6646 (obt.)

The Click procedure involved the sequential addition of azide 3 solutions (Figure 15) (DMSO solution, 10 mM, 50 equiv.), CuSO4 complex / ligand [29] (DMSO solution, 0.05 M, 20 equiv .) and TCEP (aqueous solution, 0.1 M, 40 equiv.) to a 0.2 mM solution of the corresponding ODN. The reaction mixture was stirred at room temperature overnight. The samples were then desalted by membrane, and their mass was determined by MALDI-TOF (HPA matrix)

Example 7

PCR amplification of modified dNTP DNA templates 20

7.1 PCR amplification of yeast polη

The template and primers used for PCR experiments:

Direct primer first 5’-GATTTGGGCCGGATTTGTTTC

3’-TTTTATGCTATCTCTGATACCCTTG Reverse Primer

Mold: 318 bp sequence (nt483-800 of Rad30) of yeast: 25

The 318 bp mold contains 59.75% of [A + T] and 40.25% of [C + G].

A series of commercially available thermostable polymerases were evaluated for the ability to incorporate modified deoxyuridine triphosphates 2 and 8 (Figure 10) using the PCR reaction. The DNA polymerases used were: 5

Pwo and Taq (exo-) DNA polymerase (Roche)

Vent, Vent (exo-), Therminator polymerase (from New England Biolabs) Taq (Promega).

A typical PCR reaction contained 0.05 µg of S. cerevisiae YPH 499 genomic DNA or 0.5 µg of DNA template (pDest17-Yeast polet [1-1889]), 0.3 mM of each direct and reverse primers, 1.25 U of polymerase and 10x polymerase buffer with magnesium, 200 µM of each of the unmodified dNTPs (dATP, dCTP and dGTP; 10 NEB) and 200 µM of modified dUTP. For control, natural triphosphates (dATP, dCTP, dGTP and dTTP) were used. The reactions were carried out in an overall volume of 50 µl. PCR experiments were carried out in an Eppendorf Mastercycler Personal.

For the amplification, a hot start (2 minutes at 94 ° C) was used, followed by 10 cycles of amplification (15 seconds at 94 ° C, 30 seconds at 53 ° C, 45 seconds at 72 ° C), 25 cycles (15 seconds at 94 ° C, 30 seconds at 56 ° C, 45 seconds at 72 ° C), and a final incubation for 2 minutes at 72 ° C. The PCR products were analyzed on a 2% agarose gel, staining with ethidium bromide (Figure 16). The PCR products were purified in a QIAquick (Qiagen) PCR purification kit, and used for silver staining, sequencing or digestion. The products of the PCR reaction were separated by 2% agarose gel electrophoresis containing ethidium bromide. Products were registered with an Ultra-Violet Products CCD camera. twenty

7.2 PCR amplification of polH from human

A typical PCR reaction contained 0.5 µg of DNA template (human pDest17-plH [1-2142]), 0.3 mM of each of the direct and reverse primers, 1.25 U of Pwo polymerase and 10x buffer of polymerase with magnesium, 200 µM of each of the unmodified dNTP (dATP, dCTP and dGTP; NEB) and 200 µM of modified dUTP. For control, natural triphosphates (dATP, dCTP, dGTP and dTTP) were used. The reactions were carried out in an overall volume of 50 µl. The 25 PCR experiments were carried out in an Eppendorf Mastercycler Personal.

For amplification, a hot start (2 minutes at 94 ° C) was used, followed by 10 cycles of amplification (15 seconds at 94 ° C, 30 seconds at 53 ° C, 210 seconds at 72 ° C), 30 cycles (15 seconds at 94 ° C, 30 seconds at 56 ° C, 210 seconds at 72 ° C), and a final incubation for 7 minutes at 72 ° C. The PCR products were analyzed on a 1% agarose gel, staining with ethidium bromide. The PCR products were purified in a QIAquick PCR purification kit (Qiagen), and used for digestion or sequencing.

The products of the PCR reaction were separated by 1% agarose gel electrophoresis containing ethidium bromide. Products were registered with an Ultra-Violet Products CCD camera. The results are shown in Fig. 17.

For treatment, 6 µl of 0.1 M HCl was added, and the probes were centrifuged (6000 rpm, 5 min.). Digestion was analyzed by HPLC (Interchim Interchrom Uptisphere 3 HDO column (150 x 2.1 mm), Buffer A: 2 mM TEA / HOAc in H2O; Buffer B: 2 mM TEA / HOAc in H2O: MeCN 1: 4; 0 → 30 min .; 0% → 30% B; 30 → 32 min .; 30% → 100% B; 32 → 36 min .; 100% B; 36 → 38 min .; 100% → 0% B ; 38 → 60 min .; 0% B; flow 0.2 ml / min.) (Figure 18). The different peaks were assigned by co-injection, UV and FT-ICR-HPLC-MS-MS, using the same conditions.

  40

7.3 Enzymatic digestion of PCR fragments (318 mer) incorporating an alkyne-mediated nucleotide

For enzymatic digestion, the 318 bp DNA template containing the modified nucleotide 8 (Figure 10) (approx. 10 µg in 100 µl of water) was incubated in 10 µl of Buffer A (300 mM ammonium acetate, 100 mM CaCl2, 1 mM ZnSO4, pH 5.7), 22 units of P1 nuclease (Penicillinum citrium) and 0.05 units of phosphodiesterase II from veal spleen. The sample was stirred at 37 ° C for 3 h. Digestion was completed by adding 12 µl of Buffer B (500 mM of 5 Tris-HCl, 1 mM of EDTA), 10 units of alkaline phosphatase (CIP) and 0.1 units of snake venom phosphodiesterase I (Crotalus adamanteus venom) ). The sample was stirred for another 3 h at 37 ° C.

7.4 General procedure for the triazole ligation reaction catalyzed by Cu (I) of sugar azides with modified cDNAs on polyacrylamide gels

All Click experiments were performed on 318 bp PCR fragments comprising 2 or 8 (Figure 10) 10 instead of dTTP. The modified PCR fragments were separated on 5% polyacrylamide gels, and then each gel was placed in a bath comprising 1: 1 MeOH: H2O (25 ml). A solution of sugar azide (0.1 M, 3 ml) was added to the bath, followed by a solution of CuSO4 (0.1 M, 600 µl), and finally TCEP (0.1 M, 1.2 ml) . These solutions were gently stirred at room temperature overnight.

7.5 General procedure for silver staining of sugar azides linked to double stranded DNA 15

The Click solution was decanted, and each gel was washed with 1: 1 MeOH: H2O (3 x 50 ml, 10 min. Each wash), followed by H2O (3 x 50 ml, 10 min. Each wash). Each gel was then incubated with the Tollens reagent * (400 ml) for 30 minutes. After subsequent washing with H2O (3 x 50 ml, 10 min. Each washing), the gels were developed with a developer solution comprising 100 ml of H2O, citric acid (1%, 250 µl) and formaldehyde (35%, 80 -200 µl). Depending on the concentration of formaldehyde, the development process can vary from 2 minutes to 20 minutes. twenty

* Preparation of Tollens reagent: to 80 ml of H2O AgNO3 (0.5 M, 5 ml) was added, followed by NaOH (3.0 M, 1.0 ml) and finally NH4OH (conc. NH4OH: H2O 1: 1, 2.2 ml).

conclusion

The current proposed invention involves a new and modular method for specific labeling of the nucleic acid site via the effective incorporation of modified triphosphates. These modified triphosphates comprise groups that can be further functionalized, labeled or stained, according to the needs of the user. It is also foreseeable that RNA labeling could be achieved via these methods using RNA polymerases and reverse transcriptase, respectively. Therefore, an early and effective detection of viral genomes could be performed in a host. The new protocol released about nucleic acid labeling is simple, effective, sensitive and very selective.

Example 8 (Reference) 30

Nucleic acid detection using photographic procedures

The black and white photographic procedure is one of the most sensitive chemical methods known. Photons captured on photographic paper begin the formation of small Ag nuclei, which catalyze the subsequent deposition of silver during a subsequent development procedure [22]. This results in signal amplification factors between 105 for the chemical reduction of Ag + and 1011 for the photochemical reduction of Ag +, which are similar to those obtained using PCR.

A sensitive method has been developed that uses photosensitizer dyes bound to nucleic acids, which potentially detect a particular gene at a subfemtomolar sensitivity (Figure 21). This new method is simple (you can use conventional photographic materials), fast (detection in minutes), efficient (only one biomolecule photosensitizing molecule is required) and sensitive (current non-optimized sensitivity levels are ∼10-14-40 10- 15 moles) The coupling of this methodology with gene-specific biochemical tools, such as primer extension and PCR technologies, provides a new and powerful method for the detection of nucleic acids and other analytes.

A number of fluorescent cyanine indoline (1 and 2) and quinoline (3) fluorescent derivatives were investigated for their ability to act as photosensitizers in a photographic process (Figure 22). The visible absorption maxima for 1-3 are 546 nm, 646 nm and 607 nm, respectively.

The indoline dye (1) was easily detected up to 3 x 10-14 moles (30 fmoles), while the detection limit of (2) was approximately 1-3 fmoles (Figure 22a and 22b). It was found that the detection of the quinolinic cyanine dye (3) was more sensitive (detection limit - 0.3 fmoles) than the indoline dyes (Figure 22c). A background fog may occur when radiating with intense light; however, fog can be suppressed using a low intensity infrared light source, although the sensitivity is somewhat lower (Figure 22d-f).

Control experiments (Figure 23), which use unmodified oligodeoxyribonucleotides (ODN-1, ODN-2, ODN-3), produced a small negative background photographic response with high energy irradiation at levels of 50 nmoles (i.e. difference of three orders of magnitude). Negative background levels can be completely suppressed if irradiation is carried out with an infrared lamp for 5 minutes (Figure 23b). In summary, unmodified DNA does not stain.

A series of dilutions of commercially available dyes Cy5 (λmax = 646 nm, ODN-4) and Cy5.5 (λmax = 646 nm, ODN-5) bound to the ODNs were then tested (Figure 16). It was found that the limit of detection of ODN-4 was 100 5 fmoles, while the sensitivity of the detection of Cy5.5 ODN-5 had a factor ten times greater (limit of detection 10 fmoles). Therefore, although a slight decrease in sensitivity was observed with the cyanine dye conjugated to the ODN compared to its unbound controls (Figure 24), the results clearly demonstrate that photography of biomolecules conjugated to photosensitizers, such as ODN, can be used as a very sensitive analytical tool. 10

Photosensitive dyes, for example compounds 4 or 5, can also be introduced into nucleic acids via Click chemistry (Figure 25).

conclusion

A photographic methodology can be coupled with nucleic acids linked to photosensitized dyes, to create a fast, simple, efficient and sensitive method for the detection of analytes, for example nucleic acid. fifteen

Suitable examples of photosensitizer groups are quinoline-based cyanine dyes (for example 3) or indoline dyes (for example 1 or 2), as shown in Figure 22, which offer sensitivities of at least 10 fmoles (1) , 1 fmol (2) or 0.1 fmoles (3).

Indoline-based dyes, such as Cy5 and Cy5.5, linked to oligodeoxyribonucleotides can be detected at levels of 100 fmoles and 10 fmoles, respectively. twenty

Bibliography

[1] E. Braun, Y. Eichen, U. Sivan, G. Ben-Yoseph, Nature 1998, 391, 775.

[2] M. Mertig, L. C. Ciacchi, R. Seidel, W. Pompe, A. De Vita, Nano Letters 2002, 2, 841.

[3] J. Richter, Physica E 2003, 16, 157.

[4] K. Keren, M. Krueger, R. Gilad, G. Ben-Yoseph, U. Sivan, E. Braun, Science 2002, 297, 72. 25

[5] F. Patols1v, Y. Weizmann, O. Lioubashevski, I. Willner, Angew. Chem. Int. Ed. 2002, 41, 2323.

[6] E. Katz, I. Willner, Angewandte Chemie-International Edition 2004, 43, 6042.

[7] K. Keren, R. S. Berman, E. Braun, Nano Letters 2004, 4, 323.

[8] Y. Weizman, F. Patolsky, I. Popov, I. Willner, Nano Letters 2004, 4, 787.

[9] C. F. Monson, A. T. Wooolley, Nano Letters 2003, 3, 359. 30

[10] S. Brakmann, S. Lobermann, Angewandte Chemie-International Edition 2001, 40, 1427.

[11] K. Burgess, D. Cook, Chemical Reviews 2000, 100, 2047.

[12] J. Ludwig, F. Eckstein, Journal of Organic Chemistry 1991, 56, 1777.

[13] A. Roychowdhury, H. Illangkoon, C. L. Hendrickson, S. A. Benner, Organic Letters 2004, 6, 489.

[14] B. Tollens, Chem. Ber. 1882, 15, 1635. 35

[15] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Inf. Ed. 2001, 40, 2004.

[16] B. W. Kirk, M. Feinsod, R. Favis, R. M. Kliman, F. Barany, Nucleic Acids Res. 2002, 30, 3295.

[17] X.-b. Zhong, R. Reynolds, J. R. Kidd, K. K. Kidd, R. Jenison, R. A. Marlar, D. C. Ward, PNAS 2003, 100, 11559.

[18] A. C. Syvanen, Nat. Rev. Genet. 2001, 2, 930.

[19] K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill, Q. Wang, Organic Letters 2004, 6, 4603. 40

[20] J. D. Kahl, M. M. Greenberg, J. Am. Chem. Soc. 1999, 121, 598-604.

[21] R. Micura, Angew. Chem. Int. Ed. 2002, 41 813), 2265-2269.

[22] T. H. James, in Advances in Photochemistry Vol. 13 (Eds. D. H. Volman, K. Gollnick, G. Hammond), John Wiley & Sons New York, 1986, p. 329ff.

[23] P. J. Barr, A. S. Jones, P. Serafinowski, R. T. Walker, J. Chem. Soc., Perk. Trans. 1, 1978, 10, 1263.

[24] L. Otvos, J. Szecsi, J. Sagi, T. Kovacs, Nucleic Acids Symp. Ser. 1987, 18, 125. 5

[25] K. Fujimoto, S. Matsuda, N. Takahashi, I. Saito, J. Am. Chem. Soc. 2000, 122, 5646.

[26] F. MorisVaras, X. H. Qian, C. H. Wong, J. Am. Chem. Soc. 1996, 118, 7647.

[27] D. C. M. Kong, M. von ltzstein, Carbohydrate Research 1997, 305, 323.

[28] D. Graham, J. A. Parkinson, T. Brown, J. Chem. Soc., Perkin Trans. 1, 1998, 1131-1138.

[29] Q. Wang, T.R. Chan, R. Hilgraf, V.V. Fokin, K.B. Sharpless, M.G. Finn, J. Am. Chem. Soc. 2003, 125,3192-3193. 10

Claims (78)

1.-A method to detect an analyte, selected from nucleic acids, in a sample, comprising the steps: (i) provide a sample; (ii) contacting the sample with a functionalized compound comprising at least one functional group selected from alkyne and azide groups that is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between the azide and alkyne groups that form a 1,2,3-triazole ring, and in which the functional group is attached to a nucleobase of a nucleoside or nucleotide compound or an oligomer or polymer thereof, under conditions wherein said compound forms an association product with the analyte to be detected, or its complement, 10 (iii) contacting the association product with a second reaction partner for said Click reaction, which comprises the complementary azide or alkyne group, under conditions where a Click reaction occurs between the first and second reaction couple , wherein the second reaction partner further comprises a marker group or a marker precursor group, (iv) if necessary, convert the marker precursor groups into marker groups, and (v) detect said marker groups. 2. The method of claim 1, which comprises a qualitative detection. 3. The method of claims 1 or 2, which comprises a quantitative detection. 4. The method of any one of claims 1 to 3, wherein the analyte to be detected is a nucleic acid selected from DNA and RNA. twenty 5. The method of any one of claims 1 to 4, wherein the sample is a biological sample. 6. The method of claim 5, wherein the sample is an agricultural or nutritional sample. 7. The method of claim in which the sample is a clinical sample. 8. The method of any one of claims 1 to 7, wherein the detection is carried out directly without amplification. 25 9. The method of any one of claims 1 to 7, wherein the detection is carried out in combination with an amplification step. 10. The method of any one of claims 1 to 9, wherein a detection of a sequence specific nucleic acid analyte is carried out. 11. The method of claim 10, wherein the formation of said association product comprises a sequence-specific hybridization reaction. 12. The method of any one of claims 1-11, wherein the nucleobase is selected from purine and pyrimidine bases of natural and non-natural origin. 13. The method of claim 12, wherein the functional group is attached to position 5 and 6, preferably position 5, of a pyrimidine nucleobase, or to positions 7 and 8, preferably position 7, of a nucleobase of 35 purine 14. The method of any one of claims 1 to 13, wherein the functional group is attached to the compound via a direct bond or a spacer. 15. The method of claim 14, wherein said spacer is a flexible spacer, or at least partially rigid spacer. 40 16. The method of claim 15, wherein the spacer comprises at least one group selected from alkene groups, alkyne groups, cyclic groups, particularly aromatic and heteroaromatic groups, and combinations thereof. 17. The method of any one of claims 1 to 16, wherein said functionalized compound is selected from: (i) a nucleic acid or functionalized nucleic acid analog building block, and / or (ii) a nucleic acid or functionalized nucleic acid analog. 18. The method of claim 17, wherein the formation of said association product comprises incorporating said functionalized building block into a nucleic acid or nucleic acid analog molecule. 19. The method of claim 18, wherein said nucleotide or functionalized nucleotide analog is enzymatically incorporated into a nucleic acid molecule. 5 20. The method of claim 18, wherein the formation of said association product comprises linking said functionalized nucleic acid or nucleic acid analog to the analyte to be detected. 21. The method of claim 20, wherein the binding comprises hybridization to a nucleic acid analyte. 22. The method of any one of claims 17-21, which comprises a primer extension reaction, optionally in combination with a nucleic acid amplification reaction. 10 23. The method of claim 22, comprising: provide at least one primer molecule that hybridizes, under test conditions, with the nucleic acid analyte to be detected, or its complement, and extend said primer, wherein at least one functionalized nucleotide or nucleotide analog is incorporated into the extension product. fifteen 24. The method of claim 22 or 23, comprising: provide at least one functionalized primer molecule, which hybridizes, under test conditions, with the nucleic acid analyte to be detected, or its complement, extending said primer molecule, in which an extended primer molecule is formed. twenty 25. The method of any one of claims 21-24, comprising: provide a functionalized hybridization probe, which hybridizes, under test conditions, with the nucleic acid analyte to be detected, or its complement, in which a hybridization product is formed. 26. The method of any one of claims 19-25, comprising: provide a functionalized hybridization probe, which hybridizes, under test conditions, with the nucleic acid analyte to be detected, or its complement, and subjecting the hybridization product to a treatment in which a hybridization product containing at least one mismatch is dissolved, in which the presence of an undissolved hybridization product indicates the presence and / or amount of a nucleic acid having a sequence completely complementary to the hybridization probe. 30 27. The method of claim 26, wherein the treatment comprises a digester digestion treatment. 28. The method of claim 27, wherein the unpairing digestion treatment comprises contacting the hybridization product with an unpairing glucosylase. 29. The method of claim 26, wherein the treatment comprises a differential hybridization treatment. 35 30. The method of any one of claims 1 to 29, wherein the detection is carried out by optical means. 31. The method of any one of claims 1 to 30, wherein the detection is carried out by electrical means. 32. The method of any one of claims 1 to 31, which comprises a parallel detection of a plurality of nucleic acids. 33. The method of any one of claims 1 to 32, wherein the detection is carried out on a solid surface, for example on a microarray chip. 34. The method of any one of claims 1 to 33, wherein the functional group is selected from azide groups or alkyne groups. 35. The method of any one of claims 1 to 34, wherein the second reaction pair comprises a marker group selected from optically detectable marker groups, particularly fluorescence marker groups, or from active marker groups for a redox. 5 36. The method of any one of claims 1 to 35, wherein the second reaction couple comprises a marker precursor group, selected from aldehyde groups and protected aldehyde groups, and aldehyde precursor groups. 37. The method of claim 36, wherein the conversion of the marker precursor groups into marker groups comprises forming metal depositions around the aldehyde groups. 10 38. A reagent kit for detecting an analyte in a sample, comprising: (a) a functionalized compound comprising (i) at least one Click functional group, selected from alkyne and azide groups, which is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between azide and alkyne groups which forms a 1,2,3-triazole ring, and 15 (ii) a building block of a nucleic acid or a nucleic acid analogue, in which the functional group is attached to a nucleobase, (b) a second reaction partner for said Click reaction, comprising the complementary azide or alkyne group, wherein the second reaction couple further comprises a marker group or a marker precursor group, and (c) optionally a marker forming reagent, capable of converting the marker precursor groups into marker groups. 39. Use of a reagent kit of claim 38 in a method according to any one of claims 1 to 37. 40.-Use of a compound of formula (II): C-S-N 25 in which C is a Click functional group selected from alkyne and azide groups that is a first reaction partner for a Click reaction, said Click reaction being a cycloaddition reaction (3 + 2) between azide and alkyne groups forming a ring of 1,2,3-triazole, S is a spacer, and 30 N is a nucleic acid or nucleic acid analog building block, such as a nucleoside or nucleotide compound, in which the Click functional group is attached to a nucleobase, in a method to detect a nucleic acid analyte in a sample, wherein the compound of formula (II) forms a product of association with the analyte to be detected. 41. The use of claim 40, wherein the compound of formula (II) is reacted with a reaction partner 35 comprising marker groups or marker precursor groups. 42. The use of claim 40 or 41, wherein the nucleobase is selected from purine and pyrimidine bases of natural and non-natural origin. 43. The use of claim 42, wherein the nucleobase is selected from adenine, 7-desazaadenine, guanine, 7-desazaguanine, cytosine, thymidine, uracil, inosine and xanthine. 40 44. The use of claims 42 or 43, wherein the functional group is attached to positions 5 and 6, preferably to position 5, of a pyrimidine nucleobase, or to positions 7 and 8, preferably to position 7 , of a purine nucleobase. 45. The use of any one of claims 41 to 44, wherein said spacer is an at least partially rigid spacer. Four. Five 46. The use of claim 45, wherein the spacer comprises at least one group selected from alkene groups, alkyne groups, cyclic groups, particularly aromatic and heteroaromatic groups, and combinations thereof. 47. The use of any one of claims 41 to 46, wherein the compound is a building block for the chemical or enzymatic synthesis of nucleic acids or nucleic acid analogs or a precursor thereof. 48. The use of any one of claims 41 to 47, wherein the compound is a nucleoside triphosphate, particularly a ribose nucleoside triphosphate, 2'-deoxyribose or 2 ', 3'-dideoxypribose, or a phosphoramidite, H-phosphonate or nucleoside phosphorotrieters. 49. The use of any one of claims 41 to 48, wherein the compound is a building block for the synthesis of peptide nucleic acids, morpholinic nucleic acids, or blocked nucleic acids. 50. The use of any one of claims 41 to 49, wherein the functional group is attached to the nucleobase via a spacer having a chain length of up to 10 atoms. 51. The use of claim 50, wherein the chain length is up to 3 atoms. 52. A nucleic acid molecule or a nucleic acid analog, which has at least one compound of formula (II) incorporated therein as defined according to any one of claims 40 to 51, wherein the functional group is an alkyne group attached to positions 5 and 6, preferably to position 5, of a pyrimidine nucleobase, or to positions 7 and 8, preferably to position 7, of a purine nucleobase, and in which the nucleic acid molecule or of a nucleic acid analog is capable of hybridizing with a complementary nucleic acid. 53. The nucleic acid molecule or a nucleic acid analog of claim 52, which is a nucleic acid selected from DNA and RNA, or a molecule of a nucleic acid analog selected from peptide nucleic acids, morpholinic nucleic acids or blocked nucleic acids. 54. A method for synthesizing a nucleic acid molecule or a nucleic acid analog that is capable of hybridizing with a complementary nucleic acid, which comprises incorporating a nucleotide or a nucleotide analog block comprising a compound (II ) of any one of claims 40 to 51, wherein the functional group is an alkyne group attached to positions 5 and 6, preferably to position 5, of a pyrimidine nucleobase 25, or to positions 7 and 8, preferably position 7, of a purine nucleobase, in a nucleic acid molecule or a nucleic acid analog. 55. The method of claim 54, which comprises a chemical synthesis and / or an enzymatic synthesis. 56. The method of any one of claims 54 to 55, further comprising contacting the nucleic acid molecule or a nucleic acid analog with a second reaction partner of compound (II), and carrying out a Click reaction between the first and second reaction couples, said Click reaction being a cycloaddition reaction (3 + 2) between azide and alkyne groups that forms a 1,2,3-triazole ring. 57. A nucleic acid molecule association product or a nucleic acid analog of any one of claims 52 to 53 with an analyte. 58. The product of claim 57, which is a hybridization product with a complementary nucleic acid. 35 59. Use of the method of any one of claims 1 to 37 or of the reagent kit of claim 38, for agricultural applications. 60. The use of claim 59, to detect plant nucleic acids, plant pathogens or plant pests such as insects. 61. The use of claims 59 or 60, to detect genetic variability, for example SNP in plants, 40 plant pathogens or plant pests. 62. The use of any one of claims 59 to 61, to detect or monitor resistance, tolerances or intolerances to herbicides, fungicides or pesticides, for example resistance, tolerances or intolerances to pesticides in fungi, insects or plants. 63. The use of any one of claims 59 to 62, for genotyping, for example to detect and / or differentiate 45 species or strains of fungi, insects, or plants. 64. The use of any one of claims 59 to 63, to detect and / or differentiate genetically modified organisms or strains, for example organisms or strains of fungi, insects or plants. 65. Use of the method of any one of claims 1 to 37 or of the reagent kit of claim 38, for diagnostic and forensic applications. fifty 66. The use of claim 65, to detect genetic variability, for example SNP in humans. 67. The use of any one of claims 65 to 66, to detect resistance, tolerances or intolerances, or allergies to medications. 68. The use of any one of claims 65 to 66, for genotyping. 69. The use of any one of claims 65 to 68, to detect genetically modified organisms or strains, for example organisms or strains of bacteria or viruses. 70. The use of any one of claims 65 to 69, to diagnose diseases, for example genetic diseases, allergic diseases, autoimmune diseases or infectious diseases. 71. Use of the method of any one of claims 1 to 37 or the reagent kit of claim 38, to detect the function and / or expression of genes. 10 72. Use of the method of any one of claims 1 to 37 or the reagent kit of claim 38, for brand protection. 73. The use of claim 72, to detect specific information encoded in products. 74. The use of the method of any one of claims 1 to 37 or of the reagent kit of claim 38, for nutritional applications, particularly in the area of feed. fifteen 75. The use of the method of any one of claims 1 to 37 or of the reagent kit of claim 38, for an epigenetic analysis. 76. The use of claim 75, for the analysis of a DNA methylation pattern. 77. A compound, obtainable as the reaction product of the method of claim 56. 78. An association product of the compound of claim 77 with a complementary nucleic acid. twenty