Method for detection of specific nucleic acids
The present invention relates to a method for detecting a target RNA polynucleotide by using a catcher polynucleotide which anneals to at least a portion of the target RNA polynucleotide, and a catalytically-inactive crRNA-guided (CRISPR RNA) RNA-binding protein which binds to the target RNA polynucleotide. Further envisaged is a kit for detecting a specific target RNA polynucleotide comprising inter alia one or more catcher polynucleotides complementary to at least a portion of the target RNA polynucleotide, a mature crRNA molecule which is specific for a target sequence on the target RNA polynucleotide and a catalytically-inactive crRNA-guided RNA-binding protein; as well as the use of these ingredients for the detection of the target RNA polynucleotide.
1 . A method for detecting a target RNA polynucleotide, comprising the following steps:
(i) annealing one or more catcher polynucleotides which are complementary to at least a portion of the target RNA polynucleotide to obtain a hybrid of the target RNA polynucleotide and the catcher polynucleotide, wherein the catcher polynucleotide is immobilized on a surface or connected to a bead;
(ii) annealing a mature crRNA (CRISPR RNA) molecule to a specific target sequence on the target RNA polynucleotide of the hybrid of the target RNA polynucleotide and the catcher polynucleotide to form a complex;
(iii) binding a catalytically-inactive crRNA-guided RNA-binding protein to the complex of step (ii) to obtain a protein-RNA polynucleotide complex;
(iv) removing unbound mature crRNA molecule and unbound catalytically inactive crRNA-guided RNA-binding protein;
(v) binding an antibody to the protein-RNA polynucleotide complex of step (iii); and
(vi) detecting the antibody-bound protein-RNA polynucleotide complex of step (v).
2 . The method of claim 1 , wherein the catcher polynucleotide is immobilized on magnetic beads or via a streptavidin-biotin binding.
3 . The method of claim 1 , wherein the target RNA polynucleotide is an mRNA comprising a poly A-tail.
4 . The method of claim 1 , wherein the target RNA polynucleotide comprises a poly-A-tail and the catcher polynucleotide comprises a poly T-section at least partially complementary to the poly-A-tail of the target RNA polynucleotide.
5 . The method of claim 1 , wherein the catcher polynucleotide comprises a segment at least partially complementary to a specific target sequence on the target RNA polynucleotide.
6 . The method of claim 4 , wherein the complementary section between the catcher polynucleotide and the target RNA polynucleotide comprises between 12 to 30 nucleotides.
7 . The method of claim 1 , wherein the antibody is specific for the catalytically-inactive crRNA-guided RNA-binding protein.
8 . The method of claim 1 , wherein the catalytically-inactive crRNA-guided RNA-binding protein is a Cas protein.
9 . The method of claim 8 , wherein the Cas protein is a member of the family of Cas12 or Cas13 proteins.
10 . The method of claim 1 , wherein the detection of the antibody-bound protein-RNA polynucleotide-complex of step (vi) is based on an enzymatic reaction or a non-enzymatic reaction.
11 . The method of claim 10 , wherein the enzymatic reaction is an enzymatic reaction of horseradish peroxidase (HRP), alkaline phosphatase (AP), esterase or glucose oxidase (GOD), wherein the HRP, AP, esterase or GOD is covalently linked to the antibody.
12 . The method of claim 10 , wherein the non-enzymatic reaction is a chemiluminescent reaction of an acridinium ester, wherein the acridinium ester is covalently linked to the antibody.
13 . The method of claim 1 , wherein the detection is a quantitative detection or a semi-quantitative detection.
14 . The method of claim 4 , wherein the complementary section between the catcher polynucleotide and the target RNA polynucleotide comprises 20 nucleotides.
15 . The method of claim 1 , wherein the antibody is a monoclonal antibody.
16 . The method of claim 8 , wherein the Cas protein is a Cas13 protein or a derivative thereof.
17 . The method of claim 1 , wherein the target RNA polynucleotide and/or the catcher polynucleotide comprises naturally occurring nucleotides or one or more modified nucleotides.
18 . The method of claim 17 , wherein the one or more modified nucleotides comprise oxidized nucleotides, methylated nucleotides, or labeled nucleotides.
19 . A method for detecting a target RNA polynucleotide, comprising the following steps:
(i) annealing a catcher polynucleotide to the target RNA polynucleotide to obtain a hybrid of the target RNA polynucleotide and the catcher polynucleotide, wherein the catcher polynucleotide is immobilized on a surface or connected to a bead and comprises a segment that is at least partially complementary to a portion of the target RNA polynucleotide and a poly T-section that is at least partially complementary to a poly-A-tail of the target RNA polynucleotide,
(ii) annealing a mature crRNA (CRISPR RNA) molecule to a specific target sequence on the target RNA polynucleotide to form a complex;
(iii) binding a catalytically-inactive crRNA-guided RNA-binding protein to the complex of step (ii) to obtain a protein-RNA polynucleotide complex;
(iv) removing unbound mature crRNA molecule and unbound catalytically inactive crRNA-guided RNA-binding protein;
(v) binding an antibody to the protein-RNA polynucleotide complex of step (iii); and
(vi) detecting the antibody-bound protein-RNA polynucleotide complex of step (v).