Light-Driven Activation of RNA-Guided Nucleic Acid Cleavage.

As one of the most favorable stimuli, photoactivation provides an advantageous way to manipulate biological objects. In the current study, we have successfully demonstrated the use of light activation guide RNA (gRNA) strategy for controlling CRISPR systems. By conjugating photolabile protecting groups, the CRISPR functions became minimal, but exposure of acylated gRNAs to 365 nm light triggers the removal of masking groups, leading to the rescue of CRISPR functions. Furthermore, our strategy has been successfully used to control gene editing in human cells. This proof-of-concept study therefore demonstrates the promising potential of our strategy to versatile applications in chemical biology.

Development of Visible-Light-Responsive RNA Scissors Based on a 10-23 DNAzyme.

The 10-23 DNAzyme is an artificially developed functional oligonucleotide that can cleave RNA in a sequence-specific manner. In this study, we designed a new photo-driven DNAzyme incorporating a photoresponsive DNA overhang complementary to the catalytic core region. The photoresponsive overhang region of the DNAzyme included either azobenzene components (Azos) or 2,6-dimethyl-4-(methylthio)azobenzene units (SDM-Azos) each attached to a d-threoninol linker. When the Azos or SDM-Azos were in the trans form, the photoresponsive DNA overhang hybridized with the DNAzyme, and the RNA cleavage activity was suppressed. cis Isomerization of Azos or SDM-Azos, induced by 365 or 400 nm light, respectively, destabilized the duplex between the photoresponsive overhang and the catalytic core, and the DNAzyme recovered RNA cleavage activity. Reversible photoswitching of the DNAzyme activity was achieved by use of specific light irradiation. Further, light-dependent photoswitching of protein expression in the presence of the DNAzyme was demonstrated. Thus, this photo-driven DNAzyme has potential for application as a photocontrolled gene silencing system and a photoactivatable gene expression system.

Postsynthetic Modification of DNA Phosphodiester Backbone for Photocaged DNAzyme.

Photocaged (photoactivatable) biomolecules are powerful tools for noninvasive control of biochemical activities by light irradiation. DNAzymes (deoxyribozymes) are single-stranded oligonucleotides with a broad range of enzymatic activities. In this work, to construct photocaged DNAzymes, we developed a facile and mild postsynthetic method to incorporate an interesting photolabile modification (thioether-enol phosphate, phenol substituted, TEEP-OH) into readily available phosphorothioate DNA. Upon light irradiation, TEEP-OH transformed into a native DNA phosphodiester, and accordingly the DNAzymes with RNA-cleaving activities were turned "on" from its inactive and caged form. Activation of the TEEP-OH-caged DNAzyme by light was also successful inside live cells.

In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates.

Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E. coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription in vivo. Quantitative (14)N/(15)N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly.