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 mixture comprising two or more single stranded oligonucleotide RNase inhibitors, wherein each RNAse inhibitor inhibits the activity of at least one of RNase A and RNase I, wherein the RNase inhibitors are 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 . The composition of claim 1 , wherein each oligonucleotide has a sequence corresponding to any of the oligonucleotides of SEQ ID NOs: 1-62, further comprising one or more hydrophobic nucleotide substitutions in the oligonucleotide.
3 . The composition of 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), 5-(N-tryptaminocarboxyamide)-dUTP (Trp dUTP), and Napthyl-dU.
4 . The composition of claim 1 , wherein the RNase inhibitors are in solution, immobilized on a matrix, or in powder form resulting from freeze drying or lyophilization of the inhibitor preparation.
5 . The composition of claim 4 , wherein the matrix is selected from the group consisting of beads, a reaction vessel, and a multi-well plate.
6 . The composition of claim 5 , wherein the beads, reaction vessel, or multi-well plate has a polymer surface.
7 . A method for binding at least one of RNase A and RNase I in a sample, comprising: combining the composition of claim 1 , wherein the composition is immobilized on a matrix, with a sample containing at least one of RNAse A and RNAse I.
8 . The method of claim 7 , wherein the sample further comprises RNA and the RNA is protected from degradation by an RNase.
9 . The method of claim 7 , wherein the sample further comprises a reaction mixture for synthesizing RNA.
10 . The method of claim 8 , further comprising reverse transcribing the RNA with a reverse transcriptase to form DNA and optionally amplifying the DNA for subsequent detection, diagnosis of the RNA, purification or sequencing.
11 . The method of claim 8 , wherein the RNA is obtained from a biological sample.
12 . The method of claim 8 , 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.
13 . A kit containing a matrix for receiving a sample suspected of containing an RNase, wherein the composition of claim 1 is immobilized on the matrix, and the matrix is suitable for receiving a sample containing RNAse.