Technologies & Diagnostics

Ribozymes for site-specific RNA modification provide an elegant approach for the installation of diverse functional groups, fluorophores, affinity tags, or crosslinkers at defined positions within an RNA of interest. There is increasing interest in expanding the ribozyme toolbox, since recently reported in vitro selected ribozymes have been mostly limited to labeling at adenosine sites, either by alkylation of the nucleobase or phosphodiester formation at the 2'-OH group. Here we report a cytidine-specific alkyltransferase ribozyme (CSAR) that uses O-benzylguanines as alkyl group donors. CSAR is the first ribozyme that catalyzes direct alkylation of the exocyclic amino group of a nucleobase and generates N-alkylated cytidine in a defined sequence context of a short RNA hairpin loop. In combination with tuning the electronic parameters of the transferred benzyl group, CSAR enables highly efficient cytidine alkylation for the installation of bioorthogonal functional groups.

Deoxyribonucleic acid (DNA) enables the precise arrangement and positioning of chromophores in order to study their interactions, leading, for example, to through-space energy transfer processes. BNB- and NBN-doped phenalenyls are electronically complementary fluorophores that are neutral BN/CC isosteres of the phenalenyl cation and anion, respectively. Herein, we present a pair of BNB- and NBN-doped phenalenyl-extended nucleosides, which we introduced into DNA via phosphoramidite chemistry. The two chromophores act as a donor-acceptor pair in a Förster resonance energy transfer (FRET) process, which results in the quenching of the BNB-phenalenyl fluorescence due to the nonradiative decay of the charge transfer (CT) state of the NBN-phenalenyl acceptor in an aqueous environment. The DNA duplex serves as a supramolecular scaffold to control the arrangement of the interacting BNB- and NBN-doped chromophores. The performance of the fluorophore-quencher pair was evaluated in a toehold-mediated strand displacement (TMSD) experiment, demonstrating its potential for DNA-based applications.

Synthetic nucleic acid catalysts serve as powerful tools to interrogate and manipulate biological processes with high specificity. Advances in in vitro selection, high-throughput sequencing, and computational design have yielded increasingly efficient and chemically diverse ribozymes and deoxyribozymes. Beyond applications in nucleic acid research and diagnostics, the study of nucleic acid catalysts offers fundamental insights into the origin of life and the RNA world hypothesis. This review covers both fundamental and applied perspectives and summarizes recent advancements in the field of in vitro evolution and the development of synthetic ribozymes and DNAzymes. The topics include the latest research on RNA-ligases and polymerase ribozymes as well as ribozymes and DNAzymes for targeted modification of RNA and peptides including novel catalysts for site-specific methylation, alkylation, and acylation. Finally, the versatility of RNA-cleaving DNAzymes as sensors and for the detection of RNA modifications, as well as the ability of DNAzymes to catalyze light-activating reactions are presented.