2025/10/28 by Jun Nakamura, Miyako Shiraishi, Junpei Yamamoto +1 · 1 voice
Chemistry · Biochemistry, Genetics and Molecular Biology · #Click Chemistry and Applications #CRISPR and Genetic Engineering #RNA Interference and Gene Delivery
paper · pdf · doi:10.1093/nar/gkaf1147
openalex publication_date 2025/10/28 · openalex created_date 2025/11/10 · openalex updated_date 2026/07/29
The CRISPR-Cas system revolutionized molecular biology by guiding Cas proteins to target nucleic acid sequences using customizable guide RNAs, offering unparalleled precision and versatility. Inspired by this innovation, we developed RNA-guided green fluorescent protein (RGG), a simple and programmable platform for targeting nucleic acid. Using a streamlined click chemistry approach, known for its high efficiency and specificity, we conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids, designed to complement target sequences, with azide-exposed proteins to construct RGG. Systematic optimization identified 30-nt RNA with 3'-DBCO modifications as the most effective configuration for RGG, enabling precise visualization of nuclear-localized RNAs, including NEAT1 and Satellite III RNA, in living cells. This establishes RGG as a customizable and efficient system for RNA imaging and molecular analysis, underscoring the potential of direct conjugation between guide nucleic acids and proteins to enable precise nucleic acid recognition and dynamic molecular modification in living cells.