Biology subjects

Manoharan, M.

Publications and source records attributed to Manoharan, M..

4 recordsLinked to original sources

bioRxiv2025

C-Nucleosides Stabilize RNA by Reducing Nucleophilicity at 2'-OH

Nucleotides with carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and N1-methyl-pseudouridine; these modifications were reported to slow degradation of large RNAs, but the mechanism is unknown. We measured kinetics of spontaneous and enzymatic cleavage at a single bond of synthetically modified RNAs, and find that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and of small alcohols of varied pKa suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher pKa and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/666442v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@73c194org.highwire.dtl.DTLVardef@1aa0af7org.highwire.dtl.DTLVardef@b5f8a0org.highwire.dtl.DTLVardef@d601fa_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC GraphicC_FLOATNO C_FIG SynopsisThis study reveals mechanisms by which modifications found in native and therapeutic RNAs enhance RNA stability. Carbon substitution for nitrogen or oxygen results in reduced 2'-OH nucleophilicity.

biophysics↗

bioRxiv2025

Modulation of TTR Gene Expression in the Eye using Modified Duplex RNAs

Small interfering RNAs (siRNAs) are a proven therapeutic approach for controlling gene expression in the liver. Expanding the clinical potential of RNA interference (RNAi) requires developing strategies to enhance delivery to extra-hepatic tissues. In this study we examine inhibiting transthyretin (TRR) gene expression by short interfering RNAs (siRNAs) in the eye. Anti-TTR siRNAs have been developed as successful drugs to treat TTR amyloidosis. When administered systemically, anti-TTR siRNAs alleviate symptoms by blocking TTR expression in the liver. However, TTR amyloidosis also affects the eye, suggesting a need for reducing ocular TTR gene expression. Here, we demonstrate that C5 and 2-O-linked lipid-modified siRNAs formulated in saline can inhibit TTR expression in the eye when administered locally by intravitreal (IVT) injection. Modeling suggests that length and accessibility of the lipid chains contributes to in vivo silencing. GalNAc modified anti-dsRNAs also inhibit TTR expression, albeit less potently. These data support lipid modified siRNAs as an approach to treating the ocular consequences of TTR amyloidosis. Inhibition of TTR expression throughout the eye demonstrates that lipid-siRNA conjugates have the potential to be a versatile platform for ocular drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/642595v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e9ad27org.highwire.dtl.DTLVardef@67067borg.highwire.dtl.DTLVardef@1da7eadorg.highwire.dtl.DTLVardef@1b2a552_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self

The endoplasmic reticulum aminopeptidase associated with antigen presentation (ERAAP) plays a crucial role in shaping the peptide-MHC I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we found that MCMV modulates ERAAP and induces an IFN-{gamma} producing CD8+ T cell effector response that targets uninfected ERAAP deficient cells. We also observed that ERAAP downregulation during infection led to presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulated effector markers upon MCMV infection and were sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for antiviral therapies.

immunology↗

bioRxiv2020

Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the RBD and the non-RBD domain of the spike antigen using a novel TCR-binding algorithm. A selected pool of 11 predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools containing 157 and 158 peptides both in unexposed donors and in convalescent patients suggesting that strong T-cell epitopes are likely to be missed when larger peptide pools are used in assays. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. Whether the presence of pre-existing T-cell immunity provides protection against COVID-19 or contributes to severe disease phenotype remains to be determined in a larger cohort. However, our findings raise the expectation that a significant majority of the global population is likely to have SARS-CoV-2 reactive T-cells because of prior exposure to flu and CMV viruses, in addition to common cold-causing coronaviruses.

immunology↗