In vitro selection of modified ribozymes with metal-independent RNA-cleaving activity

Ribozymes and DNAzymes are limited in their applications as nucleic acid therapeutics due to their strong dependence on metal ions. Therefore, the development of novel metal-independent ribozymes has become a key research focus. This study evaluated the fidelity of wild-type Tgo DNA polymerase in simultaneously incorporating sugar backbone-modified 2'-deoxy-2'-fluoroarabinonucleic acid (FANA) along with base-modified DNA, constructed a modified nucleotide library containing four modified nucleotides, and employed systematic evolution of ligands by exponential enrichment (SELEX) technology to select ribozymes with RNA cleavage activity. Unlike conventional methods for selecting modified functional nucleic acids, the modified library here was directly used as templates for PCR amplification, eliminating the reverse transcription step which copies the modified phenotypic functional sequence into unmodified cDNA, and dramatically simplified the screening process. This study employed an RNA substrate region composed of 15 consecutive ribonucleosides for selection. Through sequence homology analysis and secondary structure prediction, we verified the cleavage activity of the top twenty abundant sequences obtained from sequencing the most active round 4 library. A sequence denoted R4-11 with considerably high RNA cleavage activity was successfully identified, and it preferentially cleaves the phosphodiester linkage between the rC-rA dinucleotide. This study provides a method for selecting ribozymes composed of diverse modified nucleotides and offers novel tools for disease-targeted therapy through mRNA silencing.