New England Biolabs, M1284L, RNase 4

RNase 4 is a single-stranded endoribonuclease that cleaves RNA at uridine-purine (U/R) dinucleotide sites. Related Categories RNases Specification Unit Definition One unit of RNase 4 is defined as the amount of enzyme required to cleave 1.8 pmol of a 45-mer RNA substrate containing a single U/A cut site in 60 minutes at 25°C. Reaction Conditions 1X NEBuffer™ r1.1 Incubate at 37°C 1X NEBuffer™ r1.1 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7 @ 25°C) Storage Buffer 100 mM NaCl 50 mM NaOAc 200 µg/ml Recombinant Albumin 50% Glycerol pH 6 @ 25°C Heat Inactivation No FAQ Q: Does RNase 4 (NEB #M1284) cut RNA at sites other than U/A and U/G? A: The observed preference of RNase 4 is U/A > U/G >>> C/A. Low occurrence C/A activity is observed when there are high concentrations of RNase 4 relative to the target RNA. Q: How much RNase 4 (NEB #M1284) should I use to digest RNA for LC-MS/MS nucleotide sequence mapping? A: The addition of 1 µl of RNase 4 (NEB #M1284) (50,000 U/ml) yields maximal sequence coverage for 10 µg of unmodified IVT RNAs 1-5 kb in length in a 30 µl reaction (1X NEBuffer r1.1, 1 M Urea) incubated at 37°C for 1 h. However, similar to using RNase T1 or bovine pancreatic RNase A, the ideal number of RNase 4 units needed can be higher or lower based on the target RNA. RNase 4 can be diluted in 1X NEBuffer r1.1, included as a 10X solution. Q: What is the chemical identity of 5´ and 3´ oligonucleotides generated after RNase 4 (NEB #M1284) cleavage? A: Following RNase 4 cleavage, upstream 5´ oligonucleotides will end with uridine and contain a mixed population of 3´-termini, mostly 3´-phosphate or 2´, 3´-cyclic phosphate. Downstream, 3´ oligonucleotides will begin with a purine (A or G) and contain 5´-hydroxylated termini. Q: How much RNase 4 (NEB #M1284) should I use for DNA-probe directed 5´-cap mRNA analysis? A: The current recommendation is to dilute RNase 4 (50,000 U/ml) 1:30 in 1X NEBuffer r1.1. Adding 1 µl of diluted RNase 4 will be sufficient in reactions that contain 5 µg target RNA hybridized with 40 pmol of DNA probe (2023 Wolf et al. ACS Pharmacol. Transl. Sci.). Q: Can RNase 4 (NEB #M1284) be inactivated? A: Addition of 40 U of Murine RNase Inhibitor (NEB #M0314) or human placenta RNase Inhibitor (NEB #M0307) at room temperature effectively inhibits RNase 4 after standard digest conditions. For downstream applications that are not sensitive to detergents or chelating agents, a solution of 0.2 % (w/v) SDS and 20 mM EDTA pH 8.0 completely inactivates RNase 4. Q: Is RNase 4 (NEB #M1284) endonucleolytic activity sensitive to RNA modifications? A: Many uridine modifications have been tested with zero to minimal decrease in RNase 4 activity, including pseudo-, N1-methyl-pseudo-, 5-methoxy, and dihydrouridine species (Ψ, m1Ψ, mo5U, and D). To efficiently digest RNAs containing these modifications, especially m1Ψ, we recommend increasing the added volume from 1 µl to 3 µl of RNase 4 (50,000 U/ml) in equivalent 10 µg RNA digests. Since RNase 4 endonucleolytic activity requires the 2´-hydroxyl of the uridine nucleotide, modifications such as 2´ -O methyluridine (Um) completely inhibit RNase 4 activity. Q: Does RNA secondary structure impact RNase 4 (NEB #M1288) activity? A: For optimal nucleotide sequence mapping coverage, it is recommended to perform RNase 4 digests under denaturing conditions to limit the possible influence of RNA secondary structure. A digest reaction containing a final urea concentration at 1 M, incubated at 37 °C for 1 h, will ensure most RNA is available for endonucleolytic cleavage. RNase 4 endoribonuclease activity tolerates up to 3 M urea or 50% formamide (v/v). T4 Polynucleotide Kinase end repair activity tolerates up to 3 M urea or 25 % formamide (v/v). Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Q: Is magnesium required for RNase 4 activity? A: RNase 4 does not require magnesium for endoribonuclease activity.