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 method, comprising:
(a) combining an aptamer composition comprising: one or more single stranded oligonucleotides that bind to at least one RNAse selected from RNase A and RNase I,
wherein the one or more single stranded oligonucleotides are selected from:
an oligonucleotide having at least 90% sequence identity with a sequence selected from any of SEQ ID NOs: 1-62; and
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, and
wherein the one or more single stranded oligonucleotides are immobilized on a matrix;
and a sample comprising RNA and the at least one RNase,
(b) separating the immobilized oligonucleotides from the sample, thereby depleting the at least one RNase from the sample to produce a depleted sample;
(c) contacting the depleted sample with a reverse transcriptase and reverse transcribing the RNA to form DNA, and
(d) amplifying the DNA for subsequent detection, purification or sequencing.
2 . The method of claim 1 , wherein the matrix is beads.
3 . The method of claim 2 , wherein the beads are streptavidin beads.
4 . The method of claim 3 , wherein the one or more aptamers are biotinylated aptamers.
5 . The method of claim 2 , wherein the beads are magnetic beads.
6 . The method of claim 1 , wherein the sample comprises a biological sample selected from urine, saliva, blood, and serum.
7 . The method of 1 , wherein the one or more hydrophobic nucleotide substitutions are selected from 5-(N-benzylcarboxyamide)-dUTP (BndUTP), 5-(N-isobutylcarboxyamide)-dUTP (iBudUTP), 5-(N-tryptaminocarboxyamide)-dUTP (Trp dUTP), and Napthyl-dU.
8 . The method of claim 1 , wherein the DNA is amplified using LAMP or PCR.