RNase inhibitors
Compositions, methods and kits are provided that include an inhibitory oligonucleotide RNase inhibitor capable of inhibiting one or more types of RNase that coexist with biological samples or are introduced in the laboratory, thereby protecting RNA in the sample from degradation. More than one type of oligonucleotide RNase inhibitor may be combined in a mixture to inhibit a plurality of different RNases. Single oligonucleotides were identified to have inhibitory activity for a plurality of different RNases. The RNase oligonucleotide inhibitor may be immobilized on beads or other surface. It may be stored in a lyophilized form or in solution.
1. A composition comprising: a single stranded oligonucleotide RNase inhibitor that inhibits the activity of at least one of RNase A and RNase I wherein the RNase inhibitor is selected from the group consisting of:
(a) an oligonucleotide having at least 90% sequence identity with a sequence selected from any of SEQ ID NOs: 1-62; and
(b) an oligonucleotide having at least 90% sequence identity with a sequence selected from any of SEQ ID NOs: 1-62, further comprising one or more hydrophobic nucleotide substitutions in the oligonucleotide.
2. A composition according to claim 1 , wherein the oligonucleotide has a sequence corresponding to any of the oligonucleotides in SEQ ID. Nos 1-62 further comprising one or more hydrophobic nucleotide substitutions in the oligonucleotide.
3. A composition according to claim 1 , wherein the one or more hydrophobic nucleotide substitutions are selected from the group consisting of 5-(N-benzylcarboxyamide)-dUTP (BndUTP), 5-(N-isobutylcarboxyamide)-dUTP (iBudUTP), or 5-(N-tryptaminocarboxyamide)-dUTP (Trp dUTP) and Napthyl-dU.
4. The composition according to claim 1 , wherein the RNase inhibitor is in a solution, immobilized on a matrix or in powder form resulting from freeze drying or lyophilization of the inhibitor preparation.
5. The composition according to claim 4 , wherein the matrix is selected from the group consisting of a bead, a reaction vessel, or a multiwell plate.
6. A kit containing a reaction vessel for receiving a sample suspected of containing an RNase, the reaction vessel containing the composition according to claim 1 , that is either immobilized or in solution or dried wherein the dried formulation is suitable for receiving a sample containing a target RNA.
7. A method for protecting an RNA in a sample from degradation by an RNase, comprising: combining a composition according to claim 1 with a sample containing RNA or a reaction mixture for adding to or synthesizing RNA.
8. The method according to claim 7 , further comprising reverse transcribing the RNA with a reverse transcriptase to form DNA and amplifying the DNA for subsequent detection, diagnosis of the RNA, purification or sequencing.
9. The method according to claim 7 , further comprising synthesizing an RNA by means of an RNA polymerase or including an RNA polymerase in the reaction mixture for synthesizing RNA.
10. The method according to claim 7 , wherein the RNA is obtained from a biological sample.
11. The method according to claim 7 , wherein the RNA is one or more RNAs selected from the group consisting of messenger RNA, transfer RNA, ribosomal RNA, microRNA, long non coding RNA, antisense RNA, CRISPR RNA, Piwi interacting RNA, small interfering RNAs, 75K RNA, enhancer RNA, spliced leader RNA, telomerase RNA component, guide RNA, small nuclear RNA, small nucleolar RNA, ectosomal RNA, and viral RNA.