New England Biolabs supplies several Ribonucleases (RNases) for the manipulation of RNA. Endoribonucleases cleave RNA molecules in a 5´-3´ direction while exoribonucleases degrade RNA molecule in a 3´-5´ direction. The distinct activities of each RNase, including recognition of specific sequence or structural elements and reaction products, enable numerous experimental approaches.
- Exonuclease T (Exo T) (NEB #M0265) also known as RNase T, is a single-stranded RNA (1,2) or DNA (3,4) specific nuclease that requires a free 3´ terminus and removes nucleotides in the 3´→ 5´ direction. Exonuclease T can be used to generate blunt ends from RNA (5) or DNA molecules that have 3´ extensions (2) in a sequence dependent process. It can remove single-stranded primers in PCR reactions prior to DNA sequencing or SNP analysis. It can also be used to eliminate single-stranded primers for nested PCR reactions or single-stranded DNA (ssDNA) to leave behind double-stranded DNA (dsDNA) samples.
- Monarch® RNase A (NEB #T3018) is a component of the Monarch Genomic DNA Purification Kit (NEB #T3010) which degrades single-stranded RNA at C and U residues. It is used at 56°C in the Monarch Genomic DNA (gDNA) Purification Kit protocol but is active at any temperature between 20°C and 65°C.
- Ribonuclease H (RNase H) (NEB #M0297) is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA when hybridized to DNA. This enzyme does not digest single or double-stranded DNA (dsDNA). It can be used to remove poly(A) tails of mRNA hybridized to poly(dT), or to remove mRNA during second strand cDNA synthesis.
- Thermostable RNase H (NEB #M0523) specifically recognizes and cleaves the phosphodiester bonds of an RNA strand in an RNA-DNA hybrid while leaving the DNA strand intact. This thermostable nuclease exhibits the same enzymatic properties as E. coli RNase H but is active at much higher temperatures.
- Ribonuclease HII (RNase HII) (NEB #M0288) is an endoribonuclease that preferentially nicks 5´ to a ribonucleotide within the context of a DNA duplex. The enzyme leaves 5´ phosphate and 3´ hydroxyl ends (5). RNase HII prefers a dsDNA duplex containing a single ribonucleotide over an RNA/DNA hybrid substrate or an Okazaki fragment. RNase HII displays reduced hybridase activity compared to RNase H.
- RNase If (NEB #M0243) eliminates single-stranded RNA from DNA and protein preparations. It is also useful in ribonuclease protection assays. RNase If will not degrade DNA. It prefers single-stranded RNA (ssRNA) over double-stranded RNA (dsRNA).
- ShortCut® RNase III (NEB #M0245) used with its manganese-containing reaction buffer, converts long double-stranded RNA (dsRNA) into a heterogeneous mix of short (18–25 bp) interfering RNAs (siRNA) suitable for RNA interference (RNAi) in mammalian cells (6–8). It is useful for gene silencing and target validation and eliminates a trial-and-error approach with synthetic siRNA.
- XRN-1 (NEB #M0338) is a highly processive 5´→3´ exoribonuclease, requiring 5´ monophosphate and can be used in removal of RNA containing 5´ monophosphate from a RNA mixture.
References
1. Deutscher, M.P., Marlor, C.W. and Zaniewski, R. (1984) Proc. Natl. Acad. Sci. USA, 81, 4290-4293. PMID: 6379642
2 Deutscher, M.P. and Marlor, C.W. (1985) J. Biol. Chem, 260, 7067-7071. PMID: 3888994
3. Viswanathan, M., Dower, K. D. and Lovett, S. T. (1998) J. Biol. Chem, 273, 35126-35131. PMID: 9857048
4. Zuo, Y. and Deutscher, M. P. (1999) Nucleic Acid Res, 27, 4077-4082. PMID: 10497273
5. Rydberg, B. and Game, J. (2002) Proc. Natl. Acad. Sci., 99, 19954-16659. PMID: 12475934
6. Morlighem, J.E. et al. (2007) Biotechniques, 42, 599-606. PMID: 17515198
7. Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 9942-9947. PMID: 12096193
8. Calegari, F. et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 14236-14240. PMID: 12391321
