Compositions and Analysis of Dephosphorylated Oligoribonucleotides
The present disclosure relates, according to some embodiments, to compositions and analysis of RNA (e.g., dephosphorylated oligoribonucleotides) including, for example, natural and/or synthetic RNAs. A composition may comprise, for example, an endoribonuclease having an amino acid sequence that (i) corresponds to an amino acid sequence of a first species (e.g., Homo sapiens, Escherichia coli, Aspergillus oryzae, Momordica charantia, Pyrococcus furiosus, Cucumis sativus , and Sus scrofa ) or (ii) is a non-naturally occurring sequence; and/or an RNA end repair enzyme having an amino acid sequence that (i) corresponds to an amino acid sequence of a species other than the first species (e.g., a bacterial species or a bacteriophage species) or (ii) is a non-naturally occurring sequence.
1 .- 7 . (canceled)
8 . A method comprising:
(a) contacting an RNA substrate and an endoribonuclease to produce oligoribonucleotides comprising one or more unrepaired ends that are 2′,3′-cyclic-phosphorylated, 3′-phosphorylated and/or 2′-phosphorylated; and
(b) contacting an RNA end repair enzyme and the oligoribonucleotides comprising the unrepaired ends to produce oligoribonucleotides comprising one or more repaired ends that are 2′,3′-hydroxylated,
wherein the endoribonuclease specificity is why selected from (1) cleavage after a specific nucleotide followed by a purine, (2) cleavage after a specific nucleotide followed by a pyrimidine, (3) cleavage after a purine followed by a specific nucleotide, and (4) cleavage after a pyrimidine followed by a specific nucleotide.
9 . A method according to claim 8 , wherein the endoribonuclease has an average cleavage rate of the RNA substrate of once every 6-12 nucleotides.
10 . A method according to claim 8 , wherein the endoribonuclease is hRNase 4 and the RNA end repair enzyme comprises phosphodiesterase and phosphomonoesterase activities.
11 . A method according to claim 8 , wherein the endoribonuclease is selected from RNase 4, RNase T1, RNase U2, RNase A, Colicin E5, MC1, Cusativin, Csx1, MazF, ChpB, MqsR, and YafO.
12 . A method according to claim 8 , wherein the RNA end repair enzyme is a T4 polynucleotide kinase-phosphatase or a Cth polynucleotide kinase-phosphatase.
13 . A method according to claim 8 , wherein the RNA substrate is a denatured RNA substrate.
14 . A method according to claim 8 further comprising (c) characterizing the oligoribonucleotides comprising one or more repaired ends that are 2′,3′-hydroxylated, wherein the (c) characterizing optionally comprises characterizing the oligoribonucleotides comprising one or more repaired ends by one or more of gel electrophoresis, capillary electrophoresis, liquid chromatography, and mass spectrometry.
15 . A method according to claim 14 , wherein the (c) characterizing comprises separating the oligoribonucleotides from one or more of the RNA substrate, the endoribonuclease, the RNA end repair enzyme to form separated oligoribonucleotides and characterizing the separated oligoribonucleotides.
16 . A method according to claim 8 , wherein the RNA substrate comprises messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (sRNA), microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), aptamer RNA, antisense RNA, silencing RNA (siRNA), guide RNA (gRNA), or any combination thereof.
17 .- 20 . (canceled)
21 . A method comprising:
(a) contacting an RNA substrate and one or more RNA substrate binding molecules to form RNA substrate-RNA binding molecule complexes, each complex comprising a bound portion and at least one single-stranded portion, wherein each bound portion comprises at least a portion of the RNA substrate and an RNA binding molecule;
(b) contacting the RNA substrate-RNA binding molecule complexes with an enzyme composition, the enzyme composition comprising:
a single-strand-specific nucleotide-specific endoribonuclease and,
optionally, an RNA end-repair enzyme,
to form by cleavage of the RNA substrate at one or more sites within the single-stranded portion by the single-strand-specific nucleotide-specific endoribonuclease:
cleaved bound portions and
one or more fragments of the single-stranded portion;
(c) optionally separating the cleaved bound portions from the one or more fragments of the at least one single-stranded portion;
(d) optionally analyzing one or more properties of the cleaved bound portions; and
(e) optionally analyzing one or more properties of the fragments.
22 . A method according to claim 21 , wherein the single-strand-specific nucleotide-specific endoribonuclease is hRNase 4 and the RNA end repair enzyme comprises phosphodiesterase and phosphomonoesterase activities.
23 . A method according to claim 21 , wherein the endoribonuclease is selected from RNase 4, RNase T1, RNase U2, RNase A, Colicin E5, MC1, Cusativin, Csx1, MazF, ChpB, MqsR, and YafO.
24 . A method according to claim 21 , wherein the RNA end repair enzyme is a T4 polynucleotide kinase-phosphatase or a Cth polynucleotide kinase-phosphatase.
25 . A method according to claim 21 comprising the (d) optional analysis of the one or more properties of the cleaved bound portions, wherein the (d) analysis comprises characterizing at least the RNA substrate of the cleaved bound portions by one or more of gel electrophoresis, capillary electrophoresis, liquid chromatography, and mass spectrometry.
26 . A method according to claim 21 comprising the (e) optional analysis of the one or more properties of the fragments, wherein the (e) analysis comprises characterizing the fragments by one or more of gel electrophoresis, capillary electrophoresis, liquid chromatography, and mass spectrometry.
27 . A method according to claim 21 , wherein the RNA substrate comprises in vitro transcribed RNA, chemically synthesized RNA, viral RNA, prokaryotic RNA, eukaryotic RNA, archaeal RNA, or any combination thereof.
28 . A method according to claim 21 , wherein the RNA substrate comprises tissue culture RNA, biopsy RNA, feces RNA, urine RNA, lymph RNA, blood RNA, mucous RNA, sputum RNA, skin RNA, saliva RNA, wound RNA, sweat RNA, semen RNA, shoot RNA, root RNA, seed RNA, sewage RNA, sludge RNA, soil RNA, or any combination thereof.
29 . A method according to claim 21 , wherein the RNA substrate comprises messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (sRNA), microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), aptamer RNA, antisense RNA, silencing RNA (siRNA), guide RNA (gRNA), or any combination thereof.
30 . A method according to claim 21 , wherein the one or more RNA substrate binding molecules comprise a DNA probe complementary to at least a portion of the RNA substrate and wherein each bound portion comprises at least a portion of the RNA substrate and the DNA probe.