Abstract
We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure.
Highlights
RNA plays a central role in various biological processes including gene expression, gene regulation and catalysis[1,2,3,4]
Fusing the catalytic domain RNase HI with a sequence-specific zinc-finger protein resulted in an enzyme that cleaved the RNA strand in a RNA-DNA hybrid at 5 nt from the recognition sequence[5]
DNA oligonucleotide-targeted RNA cleavage provides an alternative approach for fragmentation of RNA molecules[13,14,15,16,17,18,19]
Summary
RNA plays a central role in various biological processes including gene expression, gene regulation and catalysis[1,2,3,4]. Fusing the catalytic domain RNase HI with a sequence-specific zinc-finger protein resulted in an enzyme that cleaved the RNA strand in a RNA-DNA hybrid at 5 nt from the recognition sequence[5]. This technique will require the specific nucleotide sequence to be present at the desired location of the RNA molecule. This study aimed to establish a simple ribonuclease approach for programmable site-specific RNA cleavage, based on a DNA guide and protein-DNA structure-specific recognition. Incorporating fluorescent protein to this RHAU peptide motif provided a useful probe for discrimination between different G4 topologies[39]
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