CRISPR-Cas9-mediated DNA editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage by the Cas endonuclease. Because natural gRNA is susceptible to intracellular degradation, it is desirable to chemically protect it for efficient editing. Using 4'-thioribonucleoside 5'-triphosphates and T7 transcription, we have prepared 4'-thio-modified gRNAs that guide Cas9-mediated DNA cleavage. This approach is a simple way to obtain chemically modified RNA suitable for CRISPR-Cas9 DNA editing.
CRISPR-Cas Systems , DNA Cleavage , RNA , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism
DNA and RNA are ubiquitous molecules responsible for storage and transmission of genetic information and together comprise the central dogma of molecular biology. However, the recent emergence of synthetic genetic polymers is providing an opportunity to challenge the fundamental principles of life. Herein, we describe the ongoing attempts to rewrite the central dogma with 4'-thioDNA and 4'-thioRNA, which feature a sulfur instead of an oxygen atom in the furanose ring moiety. Using reconstituted Escherichia coli gene expression machinery, studies have shown that the genetic information conserved in 4'-thioDNA can be transcribed to 4'-thioRNA and eventually translated into protein, mirroring the processes that occur in nature. Such studies underscore the feasibility of controlling life by substances other than DNA and RNA.
DNA , RNA , DNA/metabolism , Polymers/metabolism
Newly emerging or re-emerging viral infections continue to cause significant morbidity and mortality every year worldwide, resulting in serious effects on both health and the global economy. Despite significant drug discovery research against dengue viruses (DENVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), no fully effective and specific drugs directed against these viruses have been discovered. Here, we examined the anti-DENV activity of tubercidin derivatives from a compound library from Hokkaido University and demonstrated that 5-hydroxymethyltubercidin (HMTU, HUP1108) possessed both potent anti-flavivirus and anti-coronavirus activities at submicromolar levels without significant cytotoxicity. Furthermore, HMTU inhibited viral RNA replication and specifically inhibited replication at the late stages of the SARS-CoV-2 infection process. Finally, we demonstrated that HMTU 5'-triphosphate inhibited RNA extension catalyzed by the viral RNA-dependent RNA polymerase. Our findings suggest that HMTU has the potential of serving as a lead compound for the development of a broad spectrum of antiviral agents, including SARS-CoV-2.
