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:Due to their bioorthogonal properties, such as nuclease resistance, L-oligonucleotides are being increasingly employed in biomedical applications including L-aptamers, microarrays, molecular sensors, and live cell imaging probes. Given the large number of proteins that have been shown to bind nucleic acids in a nonspecific or “promiscuous” manner, it is reasonable to predict that interactions across the mirror are abundant, potentially leading to undesired effects. However how they interact with these environments and their potential consequences is not well studied. In this study, we conducted the first detailed characterization of how L-oligonucleotides interact with biological systems at the cellular level, revealing important stereochemistry-dependent behaviors of L-oligonucleotides. Notably, we show that G-rich L-oligonucleotides have potent cytotoxicity and a dramatic impact on gene expression, suggesting they make extensive interactions with endogenous proteins. In conclusion, the data presented here demonstrates that, similar to chemical modifications, stereochemistry can have a profound effect on the behavior of oligonucleotides in living systems. This work should motivate similar investigations into other modified oligonucleotides, such as XNAs, that are also perceived to be highly bioorthogonal, which may reveal unforeseen biological effects.
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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:Mirror-image proteins, composed of D-amino acids, are an attractive therapeutic modality, as they exhibit high metabolic stability and lack immunogenicity. Development of mirror-image binding proteins is achieved through chemical synthesis of D-target proteins, phage display library selection of L-binders and chemical synthesis of (mirror-image) D binders that consequently bind the physiological L-targets. Monobodies are well-established synthetic (L )binding proteins and their small size (~90 residues) and lack of endogenous cysteine residues make them particularly accessible to chemical synthesis. Here, we developed monobodies with nanomolar binding affinities against the D-SH2 domain of the leukemic tyrosine kinase BCR::ABL1. Two crystal structures of heterochiral monobody-SH2 complexes revealed targeting of the pY binding pocket by an unconventional binding mode. We then prepared potent D-monobodies by either ligating two chemically synthesized D-peptides or by self-assembly without ligation. Their proper folding and stability were determined and high affinity binding to the L-target was shown. D-monobodies were protease-resistant, showed long-term plasma stability, inhibited BCR::ABL1 kinase activity and bound BCR::ABL1 in cells and (to some extent in) cell lysates with high selectivity. Hence, we demonstrate that functional D monobodies can be developed readily. Our work represents an important step towards the possible future therapeutic use of D-monobodies when combined with emerging methods to enable cytoplasmic delivery of monobodies.
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Project description:This study examines the effect of four different image segmentation methods on the precision of the data. Keywords: other
2004-03-16 | GSE1054 | GEO
Project description:Background: Mirror-image pain (MIP), which develops from the healthy body region contralateral to the actual injured site, is a mysterious pain phenomenon accompanying many chronic pain conditions, including complex regional pain syndrome (CRPS). However, the pathogenesis of MIP still remained largely unknown. The purpose of this study is to perform an expression profiling to identify genes related with MIP in an animal model of CRPS-I. Methods: We established a rat chronic post-ischemic pain (CPIP) model to mimic human CRPS-I. RNA-sequencing (RNA-Seq), bioinformatics, qPCR, immunostaining and animal behavioral assays were used to screen potential genes in contralateral dorsal root ganglia (DRG) that may be involved in MIP. Results: The CPIP model rats developed robust and persistent MIP in contralateral hind paws. Bilateral DRG neurons did not exhibit obvious neuronal damage. RNA-Seq of contralateral DRG from CPIP model rats identified a total 527 differentially expressed genes (DEGs) vs. control rats. The expression changes of several representative DEGs were verified by qPCR. Bioinformatics analysis indicated that immune system process, innate immune response and cell adhesion were among the mostly enriched biological processes, which are all important processes involved in pain sensitization, neuroinflammation and chronic pain. We further identified DEGs potentially involved in pain mechanisms or enriched in small- to medium-sized sensory neurons or TRPV1-lineage nociceptors. By comparing with published datasets summarizing genes enriched in pain mechanisms, we sorted out a core set of genes which might contribute to nociception and pain mechanism in MIP. Conclusions: We provided by far the first study to profile gene expression changes and pathway analysis of contralateral DRG for investigating MIP mechanisms. This work may provide novel insights into understanding the mysterious mechanisms underlying MIP.
2021-09-15 | GSE174299 | GEO