Product Class: Other

RNase 4 Digestion and 3´ End Repair Mix
Gray property icon for NEB r1.1 Gray property icon for incubation temperature 37°


Catalog #M1288

Product Introduction

Graphic showing components of RNase 4 Digestion & 3´ End Repair Mix

 

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

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). 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

Diagram of product differentiation for RNase 4 and RNase 4 Digestion & 3´ End Repair Mix

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

Workflow showing use of RNase 4 or RNase 4 Digestion & 3´ End Repair Mix

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

Bar graph showing end repair formulation and relative abundance

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

Bar graph showing end repair formulation and relative abundance

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.


This product is related to the following categories:
RNases,
RNA Modification,

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)

References

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

FAQs & Troubleshooting

FAQs

  1. How much RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) should I use to digest RNA for LC-MS/MS nucleotide sequence mapping?
  2. Does RNase 4 (NEB #M1284) cut RNA at sites other than U/A and U/G?
  3. Can I use RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) for DNA-probe directed 5′ cap mRNA analysis?
  4. Can RNase 4 (NEB #M1284) be inactivated?
  5. Is RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) sensitive to RNA modifications?
  6. Does RNA secondary structure impact RNase 4 (NEB #M1288) activity?
  7. Does RNase 4 Digestion and 3′ End Repair Mix (NEB #M1288) require magnesium?
  8. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

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

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

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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