RNase 4 Digestion and 3′ End Repair Mix is a coformulation of RNase 4 (cut sites: U/A and U/G) and T4 Polynucleotide Kinase that simplifies RNase 4 digestion products into a pool of U-ending RNA oligonucleotides with a homogeneous 3′-hydroxy termini.
Generate a larger population of unique and easy-to-map oligonucleotides for improved RNA characterization by LC-MS/MS.
Endoribonuclease activity tolerates common RNA chemical modifications.
For mRNA 5´ cap analysis, use stand-alone RNase 4 with our protocol for DNA Probe-Directed Analysis of mRNA 5´Cap Structures
Product Information
RNase 4 is a single-stranded RNA endonuclease that cleaves 3´ of uridine in uridine-purine sequences (cut sites: U/A and U/G). Digestion of RNA with RNase 4 produces U-ending RNA oligonucleotides that contain cyclic 2´, 3´-phosphate and/or linear 3´-phosphate termini. RNase 4 Digestion and 3′ End Repair Mix is a coformulation of RNase 4 and the 3´ end repair activity of T4 Polynucleotide Kinase to produce a pool of U-ending RNA oligonucleotides with a 3´-hydroxy terminus. The additional 3´ end repair of RNase 4 digestion products simplifies and improves RNA sequencing coverage analysis and modification mapping by liquid chromatography-mass spectrometry (LC-MS/MS). RNase 4 endoribonuclease activity tolerates uridine base modifications such as pseudo-, N1-methyl-pseudo-, dihydro-, and 5-methoxy-uridine species (Ψ, m1Ψ, D, and mo5U).
Figure 1: RNase 4 products for comprehensive mRNA analysis by LC-MS/MS
Cap: The relative populations of 5´ mRNA ends are identified and measured using a complementary DNA probe, which blocks cleavage by RNase 4. Isolation of the cleaved product prior to LC-MS/MS analysis can be performed using a probe that contains an affinity tag. RNase 4 enables predictable, site-specific generation of 5´ end products with a simple experimental design.
Sequence: To verify the mRNA sequence and its modification status, samples are digested with RNase 4 to produce a series of defined oligonucleotides in sizes that are amenable to LC-MS/MS analysis. A coverage map can be generated by aligning the observed oligonucleotides to the reference sequence. RNase 4 Digestion and 3´ End Repair Mix simplifies mapping by producing a single pool of RNA digestion products with homogeneous 3´ hydroxy termini. One or more additional RNases (e.g., RNase T1) can be used in parallel digestions to increase the total coverage of mRNA.Tail: RNase 4 can be used to assess the mRNA 3´ end by cutting and releasing the poly(A) tail for length profiling.
Figure 2: RNase 4 Digestion and 3´ End Repair Mix simplifies RNA analysis by LC-MS/MS
Schematic of the digestion workflow with RNase 4 (NEB #M1284) or RNase 4 Digestion and 3´ End Repair Mix (NEB #M1288). The RNA sample is first heat-denatured at 90°C in the presence of 3 M urea (user supplied) for 10 minutes. After dilution to 1 M urea, RNase 4 digestion reactions are performed in 1X NEBuffer™ r1.1 provided as a 10X stock solution. The resulting oligonucleotide pool is directly analyzed by LC-MS. Boxes show the different 3´ end termini of product oligonucleotides after RNA digestion with RNase 4 versus RNase 4 Digestion and 3´ End Repair Mix.
Figure 3: 1 μl of RNase 4 Digestion and 3´ End Repair Mix in recommended digestion conditions allows optimal sequencing coverage of 20 μg RNA 1,000 to 9,000 nucleotides in length
LC-MS/MS sequencing coverage for unmodified RNA digested with RNase 4 (NEB #M1284) or RNase 4 Digestion and 3´ End Repair Mix (NEB #M1288) (please link to product). Each RNA (4,938 nucleotides or 8,944 nucleotides in length) was generated by in vitro transcription using the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040). RNase 4 Digestion and 3´ End Repair Mix is optimized for RNA digestion reactions that contain 1 µl of enzyme mix, 1 M urea (user supplied) and up to 20 µg of RNA approximately 1,000 9,000 nucleotides in length, in a 30 µl volume for 1 hour at 37°C.
Figure 4: RNase 4 Digestion and 3´ End Repair Mix is formulated for 3´ end repair of up to 20 µg of RNA at 1,000–9,000 nucleotides in length
Relative abundance of product oligonucleotide 3´ ends for RNA (4,938 or 8,944 nucleotides in length) treated with RNase 4 Digestion and 3´ End Repair Mix (NEB #1288). The co-formulated T4 PNK 3´ phosphatase activity is optimized for 30 µl RNA digestions containing 1 µl of enzyme mix, 1 M urea (user supplied) and up to 20 µg of RNA approximately 1,000–9,000 nucleotides in length, treated for 1 hour at 37°C.
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