An Unexpected Adenosine-Alkylating Ribozyme Emerged by Target Site Relocation during in Vitro Selection

Abstract

High Resolution Image Download MS PowerPoint Slide RNA-alkylating ribozymes are catalytic RNAs for post-transcriptional RNA modification that enable the site-specific installation of methyl groups, bioorthogonal functional groups, fluorophores or other tags for biochemical and biophysical analyses of RNA structures and dynamics. Here, we report new RNA-alkylating ribozymes obtained by in vitro selection in combination with high-throughput sequencing (RZ-seq) and biochemical analyses. A specific adenosine emerged as the modification site, although the structured RNA libraries provided either a cytidine or a guanosine as the prospective target nucleotide. In-line probing and comprehensive mutational profiling provided insights into the structure-activity relationship of ribozyme variants and led to a highly active R NA- a lkylating c atalytic R NA, which we termed RACR. This ribozyme uses diverse O 6 -modified guanine cofactors to transfer alkyl groups to the N 1 -position of a specific internal adenosine. The target site is located in the N A YN (N = any nucleotide, Y = C or U, target A underlined) consensus motif, and the ribozyme can be directed to diverse RNAs of interest, including tRNA and snRNA. RACR has a distinct catalytic core architecture and employs an alternative mode of RNA target engagement compared to the previously reported methyltransferase ribozyme MTR1. Thus, RACR expands the ribozyme toolbox for post-synthetic RNA modification. Moreover, the results highlight aspects of in vitro selection dynamics that have implications for future ribozyme evolution to target nucleosides other than adenosine. These efforts will also benefit from the RZ-seq method that was implemented here besides the full characterization of RACR as new and useful adenosine-modifying ribozyme.

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