RNase 4 is a single-stranded endoribonuclease that cleaves RNA at uridine-purine (U/R) dinucleotide sites.
Generate a larger population of uniquely mappable oligonucleotides for improved RNA sequence characterization by LC-MS/MS.
Endoribonuclease activity tolerates common RNA chemical modifications.
Enhance analysis of mRNA 5′ cap identity and distribution.
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). RNase 4 allows more targeted digestion of substrate RNA compared to single-nucleotide specific RNases like T1 (cut site: after G), RNase U2 (cut sites: after A and G), or bovine pancreatic RNase A (cut sites: after C and U). RNase 4 endonuclease activity tolerates uridine base modifications such as pseudo-, N1-methyl-pseudo-, dihydro-, and 5-methoxy-uridine species (Ψ, m1Ψ, D, and mo5U). Due to the chemical mechanism of RNase 4 endonucleolytic cleavage, product oligonucleotides contain heterogenous 3´ ends, where most species contain a linear 3´-phosphate or cyclic 2´,3´-phosphate.
Figure 1: RNase 4 cuts at U/A and U/G
Figure 2: RNase 4 enables comprehensive mRNA analysis by LC–MS/MS
Cap: Relative populations of 5´ mRNA ends are identified and measured with the aid of a complementary DNA probe that 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 fully 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. 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 3: RNase 4 improves mRNA sequence characterization by LC–MS/MS
A: Schematic of the digestion workflow with RNase 4. 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 reactions are performed in 1X NEBuffer™ r1.1 (NEB #M1284), provided as a 10X stock solution. The resulting oligonucleotide pool is directly analyzed by LC–MS/MS.B: Representative sequence coverage map of FLuc mRNA from two independent experiments with RNase 4 or RNase T1. HiScribe® T7 (NEB #E2040) was used to generate FLuc mRNA. Colored bars in the coverage map indicate oligonucleotides uniquely assigned to indicated positions along the reference sequence (x-axis). The percentage sequence coverage is reported.
Figure 4: Overview of the mRNA 5´ end analysis workflow with RNase 4
1: A user designed biotinylated DNA oligomer is hybridized to the 5´ end of a complementary RNA sequence upstream of a chosen UA or UG cut site. As RNase 4 is specific for single-strand RNA, any potential cut site within the hybridized DNA:RNA duplex region is protected from cleavage.2: After RNase 4 digestion, probe hybridized mRNA 5´ end products can be captured via Streptavidin Magnetic Beads (NEB #S1420). 3: Enriched DNA:RNA duplexes are eluted and analyzed by UHPLC–MS/MS
Figure 5: RNase 4 simplifies experimental design and data processing in mRNA 5´-end analysis
A: RNase 4 allows for greater specificity and flexibility in cut site selection compared to RNase H, whose cut site specificity is less predictable. Moreover, RNase H workflows require careful design and optimization of a chimeric RNA-DNA probe, where RNase 4 workflows use a DNA probe. Biotin or desthiobiotin conjugation at the 5´ or 3´ end provides the ability to enrich probe hybridized fragments by streptavidin prior to LC–MS/MS analysis.B: Representative heatmap of total FLuc mRNA 5´ end cleavage products obtained using RNase 4 or RNase H as outlined in (A) from two independent experiments.C: Estimated distribution of 5´ end-capped products and intermediates. Error bars represent the standard deviation from two independent experiments.
Figure 6: Reaction conditions for optimal RNase 4 mapping performance can be tuned based on RNA sequence and modification status
Results are plotted for RNAs of 878 (red) or 4,938 nucleotides (blue) in length. A fully m1φ substituted 4,938 nucleotide transcript (yellow) was generated by replacing UTP with cognate m1φTP during in vitro transcription with the HiScribe® T7 High Yield RNA Synthesis Kit (NEB #E2040). Similar to other RNases used for LC–MS/MS sequence mapping, the amount of RNase 4 to include for maximal sequence coverage should be empirically determined. Generally, longer and more modified RNA requires more units of enzyme for optimal mapping coverage. However, using a large excess of RNase 4, or longer incubation times does not necessarily provide the highest sequence coverage and may result in spurious digestion. RNase 4 (NEB #M1284) is provided as a 50 U/µl solution.
Product Source
An Escherichia coli strain that carries the cloned RNASE4 gene from Homo sapiens, with an N-terminal 6xHis tag.
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
M1284S
-20
RNase 4
M1284SVIAL
-20
1 x 0.05 ml
50,000 units/ml
NEBuffer™ r1.1
B6001SVIAL
-20
1 x 1.25 ml
10 X
M1284L
-20
RNase 4
M1284LVIAL
-20
1 x 0.25 ml
50,000 units/ml
NEBuffer™ r1.1
B6001SVIAL
-20
1 x 1.25 ml
10 X
Properties & Usage
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
References
Wolf et al. (2022). Human RNase 4 improves mRNA sequence characterization by LC-MS/MS. NAR. PubMedID: 35871301
Wolf et al. (2023). Selective Characterization of mRNA 5′ End-Capping by DNA Probe-Directed Enrichment with Site-Specific Endoribonucleases. ACS Pharmacol. Transl. Sci. PubMedID: 37974627
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