Biology subjects

Goddard, W. A.

Publications and source records attributed to Goddard, W. A..

3 recordsLinked to original sources

bioRxiv2026

HMCES DNA-protein cross-links promote template slippage during DNA replication

During replication, nucleolytic processing of apurinic/apyrimidinic (AP) sites in single-stranded (ss)DNA is attenuated by the evolutionarily conserved 5-hydroxymethylcytosine binding, embryonic-specific (HMCES) protein. HMCES forms a covalent thiazolidine linkage with the ring-opened aldehyde form of a ssDNA AP site to stabilize the AP site and suppress the formation of DNA double-strand breaks. The resulting HMCES DNA-protein cross-link (DPC) can then be digested by the SPRTN protease and bypassed through mutagenic translesion synthesis (TLS). Here, we use Xenopus egg extracts and molecular dynamics simulations to investigate how HMCES-DPC formation influences the mutagenicity of AP site bypass. We show that SPRTN processes the HMCES-DPC to a five amino acid peptide adduct prior to TLS. Surprisingly, the mutagenicity of HMCES-DPC bypass is insensitive to the extent of DPC proteolysis and depends only on cross-link formation, which traps the AP site in a more dynamic ring-opened configuration. We further show that the spectrum of mutations produced during bypass of HMCES-adducts strongly depends on the template strand nucleotide immediately 5 of the AP site. Our data support a model in which HMCES-DPC formation increases the conformational flexibility of the DNA template, allowing template slippage and use of the 5 template nucleotide to direct insertion opposite the adducted AP site.

biochemistry↗

bioRxiv2025

Development of conditional-siRNA programmable riboswitch for targeting adverse cardiac remodeling

Heart Failure (HF) remains a global epidemic and a significant healthcare burden, with an unmet need for novel therapies to target the preceding pathological hypertrophy in vulnerable patients. Here we report the development of novel conditional-siRNA (Cond- siRNA) constructs that are selectively activated by disease-specific RNA biomarkers to enable cell-specific inhibition of a target disease-causing RNA. We designed a Cond- siRNA that can be activated by nppa mRNA, upregulated specifically in CMs under pathological stress, to silence the key pro-hypertrophic gene calcineurin by the effector siRNA. In cellular models including neonatal rat ventricular myocyte (NRVM) and rat cardiomyocyte cell-line (H9C2), Cond-siRNA exhibited low baseline activity in the absence of the disease biomarker but achieved targeted calcineurin silencing upon nppa mRNA induction by phenylephrine (PE)-induced stress in a two-dimensional (2D) cell culture system and pressure overload in three-dimensional (3D) heart-on a chip system. NRVM transfection with the Cond-siRNA resulted in a decreased expression of calcineurin mRNA specifically after PE or pressure-overload treatment, but not after vehicle treatment, proving nppa mRNA-specific activation of the effector siRNA against calcineurin. Specificity was confirmed as Cond-siRNA did not significantly silence calcineurin in cardiac fibroblasts and T cells, lacking nppa expression. Reduced calcineurin protein levels and NFATc1 nuclear translocation correlated with decreased NRVM hypertrophy after PE treatment, confirming Cond-siRNAs efficacy. This study offers proof-of-concept for Cond-siRNA as a targeted therapy to mitigate hypertrophic progression, paving the way for novel HF treatments. One sentence summaryConditional-siRNA targeting adverse cardiac remodeling GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/633434v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1b6532borg.highwire.dtl.DTLVardef@10e3836org.highwire.dtl.DTLVardef@8c839eorg.highwire.dtl.DTLVardef@1152883_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Bystander base editing interferes with visual function restoration in Leber congenital amaurosis

Base editors (BEs) have emerged as a powerful tool for gene correction with high activity. However, bystander base editing, a byproduct of BEs, presents challenges for precise editing. Here, we investigated the effects of bystander edits on phenotypic restoration in the context of Leber congenital amaurosis (LCA), a hereditary retinal disorder, as a therapeutic model. We observed that in rd12 of LCA model mice, the highest editing activity version of an adenine base editors (ABEs), ABE8e, generated substantial bystander editing, resulting in missense mutations despite RPE65 expression, preventing restoration of visual function. Through AlphaFold-based mutational scanning and molecular dynamics simulations, we identified that the ABE8e-driven L43P mutation disrupts RPE65 structure and function. Our findings underscore the need for more stringent requirements in developing precise BEs for future clinical applications.

genetics↗