Nucleic acid beacons for fluorescent in-situ hybridisation and chip technology

Abstract

The present invention relates to beacons for fluorescent in-situ hybridisation and chip technology.

1 . A nucleic acid capable of forming a hybrid with a target nucleic acid sequence and capable of forming a stem-loop structure if no hybrid is formed with the target sequence, said nucleic acid comprising
 (a) a nucleic acid portion comprising   (a1) a sequence complementary to the target nucleic acid sequence, (a2) a pair of two complementary sequences capable of forming a stem,   (b) an effector and an inhibitor, wherein the inhibitor inhibits the effector when the nucleic acid forms a stem-loop structure, and wherein the effector is active when the nucleic acid is not forming a stem-loop structure.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the nucleic acid is suitable for in-situ hybridisation, in particular FISH. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the hybridisation takes place within a cell. 
     
     
         4 . The nucleic acid of  claim 1 , wherein the nucleic acid when covalently linked to a solid phase is suitable for hybridisation with the target nucleic acid sequence, wherein the target nucleic acid sequence is preferably provided in a cell-free sample. 
     
     
         5 . The nucleic acid of  claim 4 , wherein the target nucleic acid sequence of (at) is a nucleic acid sequence of a microorganism. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the target nucleic acid sequence of (al) is a DNA sequence or a RNA sequence, in particular a rRNA sequence. 
     
     
         7 . The nucleic acid of  claim 1 , wherein essentially the two complementary sequences of (a2) form the stem. 
     
     
         8 . The nucleic acid of  claim 1 , wherein essentially the sequence complementary to the target nucleic acid sequence of (a1) forms the loop. 
     
     
         9 . The nucleic acid of  claim 1  wherein the sequence complementary to the target nucleic acid sequence of (a1) and at least one of two complementary sequences of (a2) overlap, preferably by 1, 2, 3, 4 or 5 nucleotides or/and nucleotide analogues. 
     
     
         10 . The nucleic acid of  claim 1 , wherein the T m  of the hybrid of the sequences of (a2) is essentially equal or lower than the T m , of the hybrid of the sequence of (a1) with the target sequence, e.g. at the maximum about 5° C., about 4° C., about 3° C., about 2° C., or about 1 ° C. lower. 
     
     
         11 . The nucleic acid of  claim 1 , wherein the T m , of the hybrid of the sequences of (a2) is essentially equal or lower than the T m  of the hybrid of the sequence of (a1) with the target sequence under essentially Mg 2+  free conditions. 
     
     
         12 . The nucleic acid of  claim 1 , wherein the ΔG of the hybrid of the sequences of (a2) is smaller than 0, preferably in the absence of the target sequence. 
     
     
         13 . The nucleic acid of  claim 1 , wherein the ΔG of the hybrid of the sequences of (a2) is higher than the ΔG of the hybrid of the sequence of (a1) with a target sequence. 
     
     
         14 . The nucleic acid of  claim 1 , wherein the ΔG of the hybrid of the sequence of (a2) is lower than the ΔG a hybrid of the nucleic acid with a mismatch sequence or/and a sequence different from the target sequence. 
     
     
         15 . The nucleic acid of  claim 1 , wherein the ΔG of the hybrid of the sequence of (a1) with its target sequence is in the range of about −17 to about −25 kcal/mol under hybridisation conditions. 
     
     
         16 . The nucleic acid of  claim 1 , wherein the stem formation takes place in the presence of Mg 2+ , in particular in the presence of about 1 to about 20 mM Mg 2+ , more particular in the presence of about 5 to about 10 mM Mg 2+ , most particular in the presence of about 8 to about 10 mM Mg 2+ . 
     
     
         17 . The nucleic acid of  claim 1 , wherein the inhibitor is covalently bound to one end of the nucleic acid portion and the effector is bound to the other end of the nucleic acid portion. 
     
     
         18 . The nucleic acid of  claim 1 , wherein the effector or/and the inhibitor is coupled to the nucleic acid portion via a linker, which linker preferably comprises building blocks selected from nucleotides, nucleotide analogues, amino acids, and amino acid analogues. 
     
     
         19 . The nucleic acid of  claim 1 , wherein the inhibitor and the effector do not form part of the stem. 
     
     
         20 . The nucleic acid of  claim 1 , wherein the effector is a luminescent label, in particular is fluorescent label, and the inhibitor is a quencher. 
     
     
         21 . The nucleic acid of  claim 20 , wherein the effector and the inhibitor are suitable for fluorescence resonance transfer technology. 
     
     
         22 . The nucleic acid of  claim 1 , wherein the effector is an enzyme and the inhibitor is an inhibitor of the enzyme. 
     
     
         23 . The nucleic acid of  claim 22 , wherein the enzyme exerts an electrochemical signal. 
     
     
         24 . The nucleic acid of  claim 22 , wherein the enzyme is a reporter enzyme, preferably selected from the group consisting of tyrosinase, peroxidase, sulfite oxidase, alkaline phosphatase, glucose oxydase, guanine oxidase. 
     
     
         25 . The nucleic acid of  claim 22 , wherein the enzyme is derived from thermo- or/and hyperthermophylic organisms. 
     
     
         26 . The nucleic acid of  claim 22 , wherein the enzyme is a recombinant enzyme. 
     
     
         27 . The nucleic acid of  claim 22  wherein the enzyme is glucose oxidase and the inhibitor is an adenine nucleotide. 
     
     
         28 . The nucleic acid of  claim 1 , wherein the nucleic acid portion (a) consists of ribonucleotides, ribonucleotide analogues, deoxyribonucleotides or/and deoxyribonucleotide analogues, which nucleotide analogues are different from PNA building blocks. 
     
     
         29 . The nucleic acid of  claim 1 , wherein at least one of the two complementary sequences of (a2) comprises at least one non-matching nucleotide. 
     
     
         30 . The nucleic acid of  claim 1 , wherein the nucleic acid portion (a) is selected from the beacon sequences of Table 1. 
     
     
         31 . A combination comprising at least two nucleic acids as claimed in  claim 1 . 
     
     
         32 . The combination of  claim 31 , wherein the ΔG values of the hybrid of the sequences of (a2) or/and the hybrid of the sequence of (a1) with a target sequence of the individual nucleic acids differ at the maximum by about 4 kcal/mol. 
     
     
         33 . The combination of  claim 31 , wherein the T m  values of the hybrid of the sequences of (a2) or/and the hybrid of the sequence of (a1) with a target sequence of the individual nucleic acids differ at the maximum by about 3° C. 
     
     
         34 . The combination of  claim 31 , wherein the individual nucleic acids function uniformly under hybridisation conditions required to hybridise under in-situ hybridisation conditions. 
     
     
         35 . The combination of  claim 31 , wherein the individual nucleic acids when covalently linked to an inorganic solid phase function uniformly under hybridisation conditions required to hybridise DNA or RNA 1  wherein DNA or/and RNA are preferably provided in a cell-free sample. 
     
     
         36 . The combination of  claim 31 , wherein the individual nucleic acids when covalently linked to protein function uniformly under hybridisation conditions required to hybridise DNA or RNA, wherein DNA or/and RNA are preferably provided in a cell-free sample. 
     
     
         37 . The combination of  claim 36 , wherein the individual nucleic acids function uniformly under hybridisation conditions wherein the protein is an enzyme linked to one end and an enzyme inhibitors linked to the other end of the nucleic acid portion. 
     
     
         38 . The combination of  claim 36 , wherein the individual nucleic acids function uniformly under hybridisation conditions required to hybridise DNA or RNA, wherein the enzyme is an enzyme derived from thermo- or hyperthermophylic organisms. 
     
     
         39 . The combination of  claim 37 , where the enzyme exerts an electrochemical signal. 
     
     
         40 . A hybridisation method comprising
 (a) contacting at least one nucleic acid of  claim 1  with a biological sample,   (b) hybridising the nucleic acid or the combination of nucleic acid of (a) with the sample under conditions where the stem of the nucleic is open, and   (c) inducing conditions which allow for stem formation in those nucleic acid molecules of (a) not forming a hybrid with the sample.   
     
     
         41 . The method of  claim 40 , which is an in situ hybridisation, in particular FISH. 
     
     
         42 . The method of  claim 40 , wherein hybridisation takes place within a cell. 
     
     
         43 . The method of  claim 40 , wherein the sample comprises a microorganism to be detected. 
     
     
         44 . The method of  claim 40  wherein the target nucleic acid sequence of the nucleic acid is a nucleic acid sequence of a microorganism. 
     
     
         45 . The method of  claim 40  wherein the target nucleic acid sequence is a DNA sequence or a RNA sequence, in particular an rRNA sequence. 
     
     
         46 . The method of  claim 40  wherein the at least one nucleic acid is covalently linked to a solid phase and wherein the target nucleic acid is preferably provided in a cell-free sample. 
     
     
         47 . The method of  claim 40 , wherein step (b) comprises hybridising with a buffer which is essentially free of Mg 2+ . 
     
     
         48 . The method of  claim 40  wherein step (c) comprises washing with a Mg 2+  containing buffer. 
     
     
         49 . The method of  claim 40  wherein step (c) comprises washing at pH>8 or/and at room temperature. 
     
     
         50 . A kit comprising a nucleic acid of  claim 1 . 
     
     
         51 . A chip comprising a nucleic acid of  claim 1 . 
     
     
         52 . The chip of  claim 51  wherein the effector is an enzyme and the inhibitor is an inhibitor of the enzyme. 
     
     
         53 . The chip of  claim 51  wherein the enzyme is glucose oxidase and the inhibitor is an adenine nucleotide. 
     
     
         54 . The chip of  claim 50 , which is an electrochemical chip. 
     
     
         55 . Use of at least one nucleic acid of  claim 1  to identify the presence or absence of one or a plurality of organisms, in particular microorganisms, within a biological sample such as a plurality of live or dead matter of human, animal or/and food origin. 
     
     
         56 . Use of  claim 55  comprising ISH or/and FISH. 
     
     
         57 . Use of  claim 55 , which is a diagnostic use. 
     
     
         58 . Use of any of  claim 55 , wherein at least two nucleic acids are functioning simultaneously under identical conditions. 
     
     
         59 . A hybridisation method comprising
 (a) contacting a combination of nucleic acids of  claim 31  with a biological sample,   (b) hybridising the nucleic acid or the combination of nucleic acid of (a) with the sample under conditions where the stem of the nucleic is open, and   (c) inducing conditions which allow for stem formation in those nucleic acid molecules of (a) not forming a hybrid with the sample.