RNase HII, also known as RNase H2, is an endoribonuclease that specifically targets and severs the phosphodiester bond at the 5' end of a solitary ribonucleotide nestled within a double-stranded DNA helix, yielding a 5' phosphate and a 3' hydroxyl group (Fig 1). This enzyme exhibits minimal cleavage activity towards single-stranded RNA and is inactive against both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Functionally active within the temperature range of 50°C to 75°C, RNase HII reaches peak performance at temperatures between 70°C and 75°C. Leveraging its unique ability to perform targeted, single-site cleavage at DNA sites where a ribonucleotide is integrated, without affecting dsDNA or ssDNA, RNase HII serves as a precision "trigger" for reaction initiation, including primer activation and extension, to achieve specific amplification. This enzyme plays a pivotal role in RNase H-dependent PCR (rhPCR), loop-mediated isothermal amplification (LAMP) technology, and the degradation of RNA components in Okazaki fragments.

一. RNase H -dependent PCR(rhPCR)
The rhPCR incorporates RNase HII and specially designed, closed cleavable rhPCR primers into the PCR process. This approach leverages the unique property of RNase HII to selectively hydrolyze RNA within DNA-RNA hybrids, while leaving the phosphodiester bonds in single-stranded or double-stranded DNA and RNA intact. As a result, RNase HII only digests the DNA-RNA hybrid and allows primer extension when the primer is perfectly complementary to the target DNA sequence. This targeted action significantly enhances the reaction's accuracy. RNase HII is the sole enzyme capable of initiating the precise removal of ribonucleotides without inducing mutations by cleaving at the 5' end of ribonucleic acid. Due to its high specificity, sensitivity, and reproducibility, rhPCR offers distinct advantages in applications such as single nucleotide polymorphism (SNP) detection, gene typing, simultaneous detection of multiple targets, and environmental nucleic acid analysis. 

Technical Route

1. Primer Design

Primer design must ensure that the melting temperature after cutting is higher than the annealing temperature to ensure that the primer stably binds to the template during PCR cycles. The rhPCR primer consists of three parts:

1）5' DNA segment: Comparable in length and melting temperature (Tm) requirements to standard PCR primers, it can effectively pair with and extend from the template DNA after being cut by RNase HII enzyme.

2）A single RNA base: providing a cutting site for RNase HII.

3）3' DNA segment: A length of four or five bases with a blocker, typically a short, non-extendable molecule such as propylene glycol, which prevents the extension of DNA polymerase until the blocker is removed.

2.Cutting and Extension

At the start of the rhPCR reaction, the primer and template DNA are free. When the primer binds to the specific RNA:DNA heteroduplex template, the RNA base 5'-side of the blocked primer is recognized and cut by RNase HII enzyme, leaving a DNA oligonucleotide with a 3'-hydroxyl group, providing a starting point for DNA polymerase to extend. This is followed by the conventional PCR amplification process.

Fig 2. Diagram of the rhPCR principle ^[1]

二. Loop-mediated isothermal amplification(LAMP)

Loop-mediated isothermal amplification (LAMP) is a simple and rapid method for gene amplification that can amplify nucleic acids under isothermal conditions in a short period of time. However, due to the initiation of DNA polymerase activity during the preparation at room temperature, non-specific mismatches are formed, which can lead to a small amount of mispairing and primer dimers, causing minor contaminations that may result in false positives.
Applying RNase HII to the LAMP amplification system can effectively address the issue of false positives in LAMP technology, broadening its application in clinical diagnostics. As shown in Fig 3, the primer-activated LAMP method (PA-LAMP) using RNase HII, when the primer pairs with the target ssDNA, the RNase HII enzyme specifically cleaves the RNA on the primer, activating it and allowing primer extension, achieving specific amplification and effectively reducing false positives in the system.

YEASEN RNase HII

YEASEN's RNase HII (Cat#14539) is derived from Pyrococcus abyssi, P.a., and it is free from contaminating nucleases, nicking enzymes, and RNase residues, with low host DNA contamination, effectively reducing false positives in the system. YEASEN's RNase HII is extremely heat-resistant, maintaining its activity even after 30 minutes at 95°C, making it compatible with various rhPCR reaction systems and also suitable for LAMP and other applications.

1. E. coli genomic DNA residue < 0.5 copies/100 U

Detection of E. coli genomic DNA residue in different batches of RNase HII showed that the host genomic DNA residue of YEASEN's RNase HII is far below 0.5 copies/100 U.

Fig 4. Detection of E. coli genomic DNA residue

2. 95°C Heat Resistance Test

After heating RNase HII at 95°C for 0 to 45 minutes, the enzyme activity of RNase HII was tested. The results indicated that there was almost no loss of enzyme activity in YEASEN's RNase HII even after 45 minutes of heating.

Fig 5. 95°C heat resistance test

3. No Exonuclease., Nicking Enzyme, or RNase Residue (1 U Dose)

Incubating 1 U of different batches of RNase HII with their respective substrates DNA/RNA at 37°C for 4 hours, the results of agarose gel electrophoresis indicated that there was no exonuclease, nicking enzyme, or RNase residue in RNase HII.

Fig 6. Detection results of exonuclease, nicking enzyme, and RNase residue

Recommended Related Products

Product Application	Product Name	Catalog Number
rhPCR、LAMP	RNase HII(2 U/μL)	14539ES
rhPCR	Hieff® Taq DNA Polymerase	10101ES
RT-LAMP	Hieff® Bst Plus DNA Polymerase (40 U/μL)	14402ES
	Hifair® Ⅲ Reverse Transcriptase	11111ES

 

References

[1] Dobosy JR, Rose SD, Beltz KR, Rupp SM, Powers KM, Behlke MA, Walder JA. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol. 2011 Aug 10;11:80. doi: 10.1186/1472-6750-11-80.

[2] Du W F , Ge J H , Li J J ,et al.Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP)[J].Analytica chimica acta, 2019(1050-):1050.DOI:10.1016/j.aca.2018.10.068.