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. A composition comprising:
(a) a human RNase 4 in an amount having catalytic activity equivalent to 100 to 10,000 units of RNase T1, wherein one unit of RNase T1 causes an increase in absorbance of 1.0 at 260 nm in 15 minutes when yeast RNA is hydrolyzed at 37° C. and pH 7.5 in a reaction comprising 50 mM Tris-HCl, 2 mM EDTA, and 3 mg/mL of the yeast RNA;
(b) a bacterial RNA end repair enzyme or a bacteriophage RNA end repair enzyme; and
(c) an RNA comprising at least 1,000 nucleotides in an amount of 0.01 μg to 100 μg, wherein the human RNase 4 specificity is 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; and
wherein the RNA end repair enzyme comprises phosphodiesterase and phosphomonoesterase activities.
2. A composition according to claim 1 , wherein the human RNase 4 cleaves on average once every 6-12 nucleotides.
3. A composition according to claim 1 , wherein the RNA end repair enzyme is present in an amount of 10 units to 100,000 units.
4. The composition according to claim 1 , wherein the RNA end repair enzyme is a T4 polynucleotide kinase-phosphatase or a Cth polynucleotide kinase-phosphatase.
5. The composition according to claim 1 further comprising one or more of a denaturing agent, a buffering agent, and an RNA substrate.
6. A composition according to claim 1 , wherein the RNA 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.