Characterization of the Intracellular Behavior of Mirror Image L-Oligonucleotides Reveals the Potent Cytotoxicity of G-Rich L-RNA
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ABSTRACT:
PROVIDER: PRJNA844409 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19512937_1.fastq.gz | Fastqsanger.gz | |||
| SRR19512937_2.fastq.gz | Fastqsanger.gz | |||
| SRR19512938_1.fastq.gz | Fastqsanger.gz | |||
| SRR19512938_2.fastq.gz | Fastqsanger.gz | |||
| SRR19512939_1.fastq.gz | Fastqsanger.gz |
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Project description:Mirror-image nucleosides, as potential antiviral drugs, can inhibit virus DNA polymerase to prevent virus replication. Conversely, they may be inserted into the DNA strands during DNA replication or transcription processes, leading to mutations that affect genome stability. Accumulation of significant mutation damage in cells may result in cell aging, apoptosis, and even uncontrolled cell division. We have previously explored replicative repair of mirror-image nucleosides within Escherichia coli, and this study focuses on human cells. We constructed several plasmid substrates, each carrying a specific mirror-image nucleoside, to investigate their impact on intracellular DNA replication and transcription processes. The results showed that in HepG2 cells, L-adenosine (L-dA) was the most potent substrate in inhibiting cell replication and transcription. L-cytidine (L-dC) exhibited the highest bypass efficiency in both template and non-template strands and had the most diverse mutation types. We also observed that L-dC induced immunoregulation of the JAK-STAT signaling pathway. Therefore, our results provide a theoretical basis for the disruptions caused by mirror-image nucleosides in replication and transcription and give us some understanding that mirror-image nucleoside drugs can cause cytotoxicity.
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Project description:The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom–up liquid chromatography–tandem mass spectrometry (LC–MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC–MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC–MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.
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Project description:L-Oligonucleotides (ONs), the synthetic enantiomer of native D-nucleic acids, are being increasingly utilized for the development of diverse biomedical technologies, including molecular imaging tools, diagnostic biosensors, and aptamer-based therapeutics. Nevertheless, our understanding of how L-ONs behave in living systems falls far short of native D-ONs. In particular, despite the potential for an abundant L-ON-protein interactome, the extent to which L-ONs bind to endogenous proteins and the consequences of these interactions are unknown, posing a major hurdle towards engineering functional L-ONs with predictable intracellular behaviours. Towards closing this knowledge gap, we now report the first comprehensive characterization of L-ON–protein interactome, revealing that a wide-range nuclear proteins have the potential to bind L-RNA, especially those containing the common RNA recognition motif (RRM). Importantly, by focusing our study on cytotoxic L-RNA sequences, our data reveal key L-RNA–protein interactions that contribute to L-RNA cytotoxicity. Furthermore, we show that introducing 2-O-methyl modifications into cytotoxic L-RNA can decrease its cytotoxicity through reducing L-RNA–protein interactions, thereby demonstrating that a well-established strategy for mitigating the cytotoxic effects of antisense ONs (ASOs) can be translated across the chiral mirror. Overall, these findings greatly deepen our understanding of the intracellular behavior of L-ONs and provide valuable guidance for the future development of safe and effective L-ON-based biomedical technologies.
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