Nucleases for DNA and RNA Degradation in Research

Nucleases are enzymes that cleave phosphodiester bonds in nucleic acid molecules, degrading DNA (DNases) or RNA (RNases) in a sequence-specific, substrate-selective, or non-specific manner depending on the enzyme class - and are used for removing contaminating nucleic acids from protein and nucleic acid preparations, digesting DNA in RNA extraction, generating nicks in DNA for repair studies, and degrading the RNA template after cDNA synthesis.

MBP supplies DNases, RNases, exonucleases, and endonucleases from validated manufacturers, including ABM, with shipping across the United States, Canada, and internationally from a US base in Houston, Texas. Request a quote for nucleases for DNA and RNA degradation, cleanup, and molecular biology workflows by contacting customerservice@mbpinc.net.

Nucleases

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What Are Nucleases?

Nucleases are enzymes that cleave phosphodiester bonds between nucleotide subunits in nucleic acids. Deoxyribonucleases (DNases) act on DNA; ribonucleases (RNases) act on RNA; and some nucleases (S1 Nuclease, Mung Bean Nuclease) act on both single-stranded DNA and RNA. Nucleases are further divided into endonucleases, which cleave internal phosphodiester bonds, and exonucleases, which progressively remove nucleotides from the 5' or 3' terminus of a strand. Major research nucleases include DNase I (non-specific endonuclease for both ss- and ds-DNA), RNase A (cleaves single-stranded RNA at pyrimidine residues), RNase H (degrades the RNA strand of RNA: DNA hybrids), Exonuclease III (removes nucleotides from 3'-recessed or blunt ends of duplex DNA), S1 Nuclease (degrades single-stranded DNA or RNA), and RNase R (degrades linear RNA but not circular RNA). Match the nuclease to the substrate type (single-stranded vs. double-stranded, DNA vs. RNA), the directionality required (endo vs. exo), and cofactor compatibility with your reaction buffer.

What you will find:

PureRec RNase A (Recombinant): A ribonuclease free of animal-derived components, designed for the swift elimination of RNA from DNA samples, ensuring no threat of viral contamination.
DNase I Set (RNase-free): A highly effective endonuclease formulated for complete DNA digestion in delicate RNA applications while preserving the integrity of the transcript.
PureRec Duplex-Specific Nuclease (DSN): A unique enzyme designed to equalize high-abundance transcripts, greatly enhancing the identification of rare variants in cDNA libraries.
RNase R: A distinctive 3'-to-5' exoribonuclease that breaks down linear RNA but preserves circular RNA forms, crucial for the identification of circRNA.
RNaseOFF Inhibitor: A strong protein-based barrier that counteracts a wide range of RNases, maintaining the delicate transcriptional environment of your samples.

How to Choose Nucleases

DNase selection

DNase I (bovine pancreatic, recombinant) non-specifically cleaves both single- and double-stranded DNA in the presence of Mg2+ (random cleavage) or Mn2+ (double-strand cuts at approximately the same position on both strands). Use RNase-free DNase I for RNA preparations to remove genomic DNA contamination. Use Plasmid-Safe ATP-Dependent DNase to selectively degrade linear double-stranded DNA while preserving supercoiled and relaxed circular plasmid DNA. Baseline-ZERO DNase is optimized for the complete removal of trace DNA without damaging RNA.

RNase selection

RNase A cleaves single-stranded RNA at pyrimidine (C and U) residues to produce 3'-pyrimidine nucleotides; it is used to remove RNA from DNA preps, to perform RNase protection assays, and to analyze RNA secondary structure. RNase H degrades the RNA strand of RNA: DNA duplexes and is used after first-strand cDNA synthesis to remove the mRNA template. RNase R (3'->5' exoribonuclease) degrades all linear RNA without affecting circular RNA, and is used in circular RNA enrichment workflows and to characterize circular RNA splice junctions. Terminator 5'-Phosphate-Dependent Exonuclease (Terminator) digests linear RNA with a 5'-monophosphate, enriching for capped mRNA or circular RNA.

Exonuclease selection

Exonuclease III (Exo III) removes nucleotides from the 3' terminus of duplex DNA and generates single-stranded regions used in unidirectional nested deletions, mapping regulatory elements, and generating single-stranded templates for sequencing. Exonuclease I (Exo I) digests single-stranded DNA in the 3'->5' direction and is used after PCR to remove unincorporated primers before Sanger sequencing. Lambda Exonuclease removes nucleotides from 5'-phosphorylated duplex DNA in the 5'->3' direction and is used in isothermal amplification and strand displacement workflows.

Cofactor and inhibitor sensitivity

DNase I requires Mg2+ or Ca2+ for activity and is completely inhibited by EDTA at a 5 mM concentration. RNase A is active across a wide pH range (optimum 7.0-7.5) and is exceptionally stable; inactivation requires proteinase K digestion, phenol-chloroform extraction, or RNase inhibitor protein. RNase H requires Mg2+; RNase R requires Mg2+ and is most active between 37-50 degrees C.

Purity and application grade

For RNA-focused applications, all nucleases used on DNA substrates must be certified RNase-free. For DNA-focused work, DNase-free grades of RNases are required. Confirm lot-specific QC contamination data from the manufacturer before use in sensitive workflows.

Specifications Context

DNase I activity is expressed in Kunitz units: 1 unit increases A260 by 0.001 per minute per mL during DNA hydrolysis at pH 5.0. RNase A activity is expressed as the amount of enzyme hydrolyzing 50% of an RNA substrate under defined conditions; it remains active at a wide range of temperatures and is notoriously difficult to inactivate (requires boiling in SDS, proteinase K digestion, or phenol-chloroform extraction). RNase R from E. coli degrades linear RNA processively in the 3'->5' direction and is used at 2-10 U per microgram of total RNA to enrich for circular RNA species. RNase R treatment before library construction is the standard method for circular RNA sequencing (circRNA-seq) workflows on both short-read and long-read platforms. For protecting RNA from RNase contamination during nuclease-adjacent workflows, see RNase inhibitors in the parent Enzymes and Inhibitors category.

Safeguard your molecular images—reach out to the MBP team now to obtain a quote for our sophisticated nucleases and inhibitors.

FAQ

DNase I is an endonuclease that cleaves both single-stranded and double-stranded DNA to yield 5'-phosphorylated and 3'-hydroxylated oligonucleotides. In the presence of Mg2+, cleavage of each strand occurs independently at random positions; in the presence of Mn2+, both strands are cleaved at approximately the same site. DNase I is used to remove genomic DNA from total RNA preparations, to introduce random nicks in DNA for nick translation, to perform DNase I footprinting, and to generate fragmented DNA libraries for sequencing.

Endonucleases cleave phosphodiester bonds within a polynucleotide chain, generating internal cuts and fragments of variable length. DNase I, RNase A, and restriction enzymes are endonucleases. Exonucleases remove nucleotides one at a time from either the 5' or 3' terminus of a DNA or RNA strand. Exonuclease I degrades ssDNA from the 3' end; Lambda Exonuclease removes from the 5' end of duplexed DNA; RNase R degrades linear RNA from the 3' end. Some enzymes (S1 Nuclease) have both endo- and exonuclease activities depending on substrate structure.

RNase A is the standard reagent for removing RNA contamination from DNA preparations. Add 20-50 microgram per mL of RNase A to the cell lysate or DNA solution, incubate at 37 degrees C for 30-60 minutes, then inactivate by proteinase K digestion (100 microgram per mL, 37 degrees C, 30 minutes) or phenol-chloroform extraction. DNase-free-certified RNase A is required to avoid introducing DNA-degrading contamination into the preparation. For plasmid miniprep protocols, RNase A is typically included in the resuspension buffer at 50-100 microgram per mL.

RNase R is a 3'->5' processive exoribonuclease from E. coli that degrades linear RNA molecules of any sequence but cannot digest circular RNA due to the absence of a free 3' terminus. Treatment of total RNA with 5-20 U of RNase R per microgram RNA at 37 degrees C for 30-60 minutes depletes linear mRNA, rRNA, and tRNA while enriching for circular RNA (circRNA) species. RNase R-treated RNA is then used as input for circRNA-seq library preparation on Illumina, ONT, or PacBio platforms to identify and quantify backsplice junctions.

DNase I used for RNA-seq sample preparation must be RNase-free grade and must be inactivated before cDNA synthesis or library preparation. Heat inactivation is performed by adding EDTA to 2.5 mM and heating to 75 degrees C for 10 minutes; the EDTA chelates the Mg2+ required for DNase I activity without damaging RNA. Alternatively, column-based RNA cleanup removes DNase I and reaction components simultaneously. Failure to inactivate DNase I will result in degradation of cDNA produced in the subsequent reverse transcription step.

Exonuclease III (Exo III) from E. coli catalyzes the stepwise removal of mononucleotides from 3'-hydroxyl termini of double-stranded DNA in the 3'->5' direction, generating single-stranded DNA tails. It acts on blunt ends and 5' overhangs but not on 3' overhangs, which are resistant. Exo III is used to create unidirectional nested deletion series for mapping promoter or regulatory element boundaries, to generate single-stranded sequencing templates, and in Gibson Assembly workflows to create compatible single-stranded regions for annealing.

S1 Nuclease from Aspergillus oryzae specifically degrades single-stranded DNA and RNA under low-pH conditions (optimum pH 4.0-4.3) in the presence of Zn2+, while double-stranded DNA is highly resistant. DNase I cleaves both single- and double-stranded DNA at neutral pH in the presence of Mg2+. S1 Nuclease is used for removing single-stranded tails or hairpin loops from duplex DNA, analyzing RNA secondary structure by S1 mapping, and processing heteroduplex molecules. Its activity on double-stranded DNA increases at very high enzyme concentrations or elevated temperatures.

Exonuclease I (Exo I) combined with Shrimp Alkaline Phosphatase (SAP) is the standard enzymatic cleanup method for removing unincorporated primers and dNTPs from PCR products before Sanger sequencing. Exo I degrades single-stranded DNA (primers) in the 3'->5' direction; SAP dephosphorylates unincorporated dNTPs. The ExoSAP-IT or equivalent cleanup protocol uses both enzymes at 37 degrees C for 15-30 minutes followed by heat inactivation at 80 degrees C for 15 minutes. This approach is faster and lower-cost than column-based cleanup for single-tube reactions.

MBP supports bulk purchasing of nucleases including DNase I, RNase A, RNase H, and exonucleases for high-throughput genomics labs, core facilities, and institutional buyers. MBP is a registered vendor for Howard Hughes Medical Institute, Vanderbilt University, and MD Anderson Cancer Center, and accepts institutional purchase orders. Submit bulk inquiries through the Quick Order portal at mbpinc.net or contact MBP directly for volume pricing and delivery scheduling.

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