RH2Fusion: A universal tool for precise DNA fragment assembly.

Despite its limitations, restriction enzyme (RE)-mediated cleavage remains the prevalent method for generating sticky ends in DNA assembly. Here, we present RNase HII Fusion (RH2Fusion), a robust system for user-defined sticky ends, enabling scarless assembly of multiple DNA fragments alongside simultaneous site-directed mutagenesis (SDM) at multiple sites. In bacterial cells, DNA fragments with ribonucleotide modifications are expected to form complementary 3' overhangs after RNase HII treatment, followed by annealing and recombination via the bacterial self-repair system. In vitro, RNase HII-mediated cleavage produces similar overhangs, which are subsequently processed and ligated by YgdG and T4 DNA ligase, enabling efficient DNA assembly. We report for the first time that Escherichia coli Exonuclease IX (YgdG) possesses ribonuclease-specific cleavage activity, selectively cleaving ribonucleotides without cleaving deoxyribonucleotides. Through the fusion of RNase HII and YgdG, novel constructs RNase RY (RNase HII-YgdG) and RNase YR (YgdG-RNase HII) are generated, each showcasing dual enzyme functionality. In conclusion, RH2Fusion offers a rapid, effective, and versatile alternative for DNA assembly, empowering researchers across diverse fields like synthetic biology and genetic engineering. This transformative tool is poised to significantly enhance the capabilities of DNA manipulation and advance molecular biology research.