Product Class: Other

RNase 4
Gray property icon for NEB r1.1 recombinant Gray property icon for incubation temperature 37° Gray property icon for heat inactivation no

Product Introduction

Mechanism of RNase 4

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.
  • For simplified analysis and enhanced sensitivity, RNase 4 with 3´ End Repair is available for LC-MS/MS applications

Bioz Badge Exists : True
Catalog # Size Concentration
M1284S 2500 units 50000 units/ml
M1284L 12500 units 50000 units/ml

Product Information

Description

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

Mechanism of RNase 4



Figure 2: RNase 4 enables comprehensive mRNA analysis by LC–MS/MS

Overview of RNase 4 role in analysis via LC-MS for mRNA cap, sequence and poly(A) tail

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

Protocol for in silico digestion with RNase 4, and coverage results for RNase 4 as comared to RNase T1


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

RNase 4 workflow: Digestion, enrichment and analysis

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

Diagram of RNase 4 digestion with biotinylated DNA probe or biotinylated DNA-RNA chimeric probe, and analysis of product length and fraction intensity of RNase 4 as comared to RNase H


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

Line graph depicting coverage for unmodified RNA at various lengths, with or without modification

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:
RNases,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M1284S -20
    Component Name Component # Stored at (°C) Amount Concentration
    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
  • NEB # Stored at (°C)
    M1284L -20
    Component Name Component # Stored at (°C) Amount Concentration
    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

Materials Required but not Supplied

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

References

  1. Wolf et al. (2022). Human RNase 4 improves mRNA sequence characterization by LC-MS/MS. NAR. PubMedID: 35871301
  2. 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

FAQs & Troubleshooting

FAQs

  1. Does RNase 4 (NEB #M1284) cut RNA at sites other than U/A and U/G?
  2. How much RNase 4 (NEB #M1284) should I use to digest RNA for LC-MS/MS nucleotide sequence mapping?
  3. What is the chemical identity of 5´ and 3´ oligonucleotides generated after RNase 4 (NEB #M1284) cleavage?
  4. How much RNase 4 (NEB #M1284) should I use for DNA-probe directed 5´-cap mRNA analysis?
  5. Can RNase 4 (NEB #M1284) be inactivated?
  6. Is RNase 4 (NEB #M1284) endonucleolytic activity sensitive to RNA modifications?
  7. Does RNA secondary structure impact RNase 4 (NEB #M1288) activity?
  8. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?
  9. Is magnesium required for RNase 4 activity?

Quality, Safety & Legal

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Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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