RapiZyme RNases

RapiZyme RNases

Tunable RNA digestion for streamlined LC-MS sequencing

Tunable RNA digestion for streamlined LC-MS sequencing

CRISPR sgRNA and mRNA require vigorous characterization to confirm their identity, sequences, and purity. These RNA molecules are prime candidates for LC-MS analysis due to their complex functionality, modifications, and variants present. Because of their size, digestion with an endonuclease such as an RNase is often performed prior to injection. The resulting digestion products can be readily detected using ion pairing reversed phase (IPRP) or hydrophilic chromatography (HILIC) coupled with mass spectrometry (MS).

Waters RapiZyme MC1 and RapiZyme Cusativin offer controlled, reproducible RNA cleavage at a range of dinucleotide sites. RapiZyme RNases produce a variety of unique RNA fragments with complementary overlap. For LC-MS users, this gives tunable digestions that improve confidence in peak identifications with a result to increase sequence coverage and molecular insights. Each tube includes 10,000 units of RapiZyme RNase, which is enough enzyme reagent to complete up to 100 digestion reactions of 10 µg RNA samples.

  • Achieve maximum sequence coverage for mRNA and sgRNA
  • Work with RNases specifically designed for LC-MS analysis
  • Eliminate the need for denaturants and RNases inhibitors when performing digestion
  • Achieve cleaner signal and sensitivity with cyclic phosphate products
  • Accelerate the development and analysis of RNA therapeutics
RapiZyme MC1 and RapiZyme Cusativin lyophilized enzyme -10,000 Unit vials. RapiZyme RNases

Specifications

Overview

  • Enhance results with more complete sequence coverage for LC-MS characterization of RNA
  • Improve efficiency with high purity, highly active enzyme reagents up to 100 reactions depending on substrate
  • Simplify your analysis using a ready-to-automate protocol for digestion
  • Avoid over digestion and instrument/column contamination with reliable heat inactivation that does not require additional inhibitor reagents
  • Target uridine or cytidine residues using two enzymatic specificities
  • Maximize sensitivity and simplify data interpretation with enzyme digestion properties integrated within waters_connect MAP Sequence
  • Pair with IonHance HFIP mobile phase additive for a 2x decrease in unwanted sodium and potassium adducts

Recommended Use: For improved sequence coverage for CRISPR sgRNA and mRNA therapeutics.


A quest to digest

RapiZyme MC1 and RapiZyme Cusativin have reproducible cleavage specificities. Double labeled, quenched RNA substrates have been applied to study RapiZyme MC1, and RapiZyme Cusativin. Upon cleavage by the RNase, a FAM-labeled test substrate is separated from its Black Hole Quencher conjugate. The digest event is then detected using absorbance-based techniques providing a quantitative measurement for on-target cleavage. A negative control, HEX-labeled substrate (e.g. polyA) is additionally applied to evaluate the off-target cleavage. The ratio of FAM/HEX provides insights into the activity of each enzyme and can be performed under various conditions including temperature, pH, buffer composition, enzyme units, and time. These analyses were performed to determine the optimal reaction conditions for RapiZyme Cusativin and RapiZyme MC1.


Let’s get specific

RapiZyme MC1 and RapiZyme Cusativin are high purity, highly active RNases that offer complementary cleavage specificities. These recombinant, tunable enzymes are designed to accelerate the development and analysis of RNA therapeutics by facilitating controlled digestion of synthetic oligonucleotides, mRNA, and sgRNA. RapiZyme MC1 exhibits its most selective, efficient digestion rates at [A/U/C]pU dinucleotide bonds at pH 8, while RapiZyme Cusativin cleaves most efficiently and selectively at Cp[U/A/G] dinucleotide sequences at pH 9. Parameters such as temperature, pH, and enzyme to substrate ratio may be altered to optimize for sample specific effects.


Cover your bases

RapiZyme MC1 and RapiZyme Cusativin exhibit a preference for substrate docking and cleavage at specific dinucleotide locations. RapiZyme MC1 has a tendency for cleaving at the 5’ end of uridine and RapiZyme Cusativin at the 3’ end of cytidine. Both enzymes demonstrate controlled, repeatable cleavage products. The idiosyncratic nature of these RNases results in partial digestion, generating longer products in addition to unique and overlapping fragments. Partial digestion, providing a plethora of information, creates more diverse fragments to investigate for structural elucidation. Data interpretation can be expedited with informatics tools such as waters_connect MAP Sequence, which predicts in silico fragments (mRNA Cleaver) based on the known cleavage properties of the chosen enzyme and maps the observed data to the sequence of the RNA molecule being investigated.


Resources

Documents

Documents


Support

Support


Related

Analyze gene therapeutics to learn about the potency and safety of your sample with Waters Gene Therapeutics (GTx) Size-Exclusion Chromatography (SEC) Columns, designed to provide accurate, high-throughput data.

Develop and qualify methods confidently with bespoke Oligonucleotide and Nucleic Acid Standards for LC and LC-MS analysis.

Reliable and robust critical quality attribute analysis of aggregates for Lipids nanoparticles (LNPs), nucleic acids, and viral vectors using GTxResolve columns.

Upgrade your lab workflow to accommodate oligo quantification, characterization and impurities analysis with Waters oligonucleotide reversed-phase columns and standards.

Develop high resolution, ion-pairing free analysis methods for synthetic oligonucleotides, digested nucleic acids and capsid proteins with GTxResolve Premier BEH Amide Columns.

Offers and Discounts

Back To Top Back To Top

RapiZyme RNases are advanced enzymes designed to improve RNA analysis by creating precise and consistent RNA fragments. Unlike traditional RNA-cleaving enzymes, these specially engineered endonucleases cut RNA at specific locations, generating fragments of optimal length for liquid chromatography-mass spectrometry (LC-MS) analysis. This precision makes them ideal for RNA sequencing and modification mapping, where detailed and accurate information is crucial.

One of the primary advantages of RapiZyme RNases is their ability to simplify the study of RNA modifications, such as methylation and other chemical changes. These modifications play a significant role in RNA function and regulation, and identifying their locations provides valuable insights into RNA biology. By producing uniform RNA fragments, RapiZyme RNases improve sequence coverage and make it easier to detect and analyze these modifications.

These enzymes are highly versatile and compatible with various RNA sources, including complex biological samples and RNA-based therapeutics like mRNA vaccines. This adaptability makes them valuable in diverse applications, such as transcriptome analysis, epitranscriptomics, and quality control for RNA medicines. For instance, in RNA therapeutics, they help ensure that molecules are intact and properly modified, which is critical for safety and effectiveness.

RapiZyme RNases also streamline workflows by reducing the need for extensive preprocessing steps, saving researchers time and effort. Their ability to generate high-quality, reliable data supports both basic research and applied biotechnology, such as biomarker discovery and the development of RNA-based diagnostic tools.

In summary, RapiZyme RNases are cutting-edge tools that enhance RNA research and analysis. They provide precise, reproducible results, simplify complex workflows, and open up new possibilities for studying RNA and developing RNA-based technologies. These enzymes are essential for advancing our understanding of RNA biology and improving the development of RNA therapeutics and diagnostics.