Biology subjects

Zain, R.

Publications and source records attributed to Zain, R..

6 recordsLinked to original sources

bioRxiv2026

Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats

Expansions of the pathogenic (AAGGG)n repeat within the RFC1 gene cause the hereditary disease CANVAS. We have shown that this repeat impedes DNA replication both in vitro and in vivo. Here, we investigated the mechanisms of replication fork stalling and reversal at (AAGGG)n repeats within an SV40-based episome in human cells. A comprehensive 2-dimensional electrophoretic analysis of replication intermediates revealed a strict orientation dependence of the replication block, evident only when the homopurine sequence was in the lagging-strand template. Fork accumulation was further increased in the presence of the G4-DNA stabilizer pyridostatin, implicating G4-DNA in the process. This stalling was followed by an accumulation of reversed forks and further analysis revealed roles for SMARCAL1 and RAD51 in this process. Biochemical studies showed that SMARCAL1, with assistance from RPA, efficiently remodels a fork carrying the G4-forming AAGGG repeat in the ssDNA gap, but not a substrate carrying the repeat in triplex conformation. We conclude that stalling results from G4-DNA formation by the repeat, which then triggers SMARCAL1-mediated fork reversal at the repeat. Given the mirror symmetry of the sequence, the nascent strands at the reversed forks likely adopt a triplex conformation, leading to a thorough accumulation of branched structures.

molecular biology↗

bioRxiv2026

Enhancing the efficacy of siRNA Antibody Oligonucleotide Conjugates (AOCs) through chemical design

Antibody-oligonucleotide conjugates (AOCs) offer a promising solution to delivery challenges of therapeutic oligonucleotides. However, the relationship between their complex chemical architectures and biological activity remains poorly understood, limiting the development of important structure-function relationships. For siRNA-containing AOCs, increasing the siRNA-to-antibody ratio beyond one (drug-to-antibody ratio, DAR>1) reduces potency, attributed to altered pharmacokinetics arising from increased negative charge density. Here, we investigated whether simple chemical modifications could improve AOC efficacy and mitigate limitations associated with higher DAR. Introduction of a single C16 lipid modification to the siRNA significantly enhanced target gene silencing compared to the unmodified AOC. Extending this modification to a DAR2 architecture, in which two siRNAs are conjugated per antibody, restored the loss of activity associated with increasing DAR from 1 to 2. Notably, at an equivalent siRNA dose (1 mg/kg), the DAR2-C16 AOC requires half the amount of antibody while achieving knockdown comparable to the DAR1-C16 AOC. We also developed a charge-balancing ionizable linker (CBIL) designed to partially compensate for the negative charge of siRNA. Incorporation of the CBIL enhanced target gene silencing in heart and skeletal muscle without a corresponding increase in hepatic activity, resulting in a shift toward greater extrahepatic activity relative to liver. Together, these findings demonstrate that chemical modification of both the siRNA payload and antibody-siRNA linker can be used to tune AOC potency and tissue activity, while enabling higher payload loading without compromising efficacy. These results establish chemical design as an important strategy for expanding the architecture and therapeutic potential of AOCs.

biochemistry↗

bioRxiv2026

Lipid-ASO therapeutics exhibit differential tissue targeted delivery upon systemic or local CNS administration

Antisense oligonucleotides (ASOs) are a powerful therapeutic modality, but their full potential is hindered by pharmacokinetic properties that affect tissue and cellular delivery. Lipid conjugation is increasingly used to modulate ASO's biodistribution and promote extrahepatic activity, yet lipid dependent effects on in vivo functional delivery, particularly in the central nervous system (CNS), remain less explored. Here, we performed a side by side in vivo comparison of cholesterol, palmitic acid (C16:0), docosanoic acid (C22:0), and eicosapentaenoic acid (C20:5) conjugated to a fully phosphorothioated 3 10 3 LNA gapmer ASO targeting the Malat1 long non coding RNA. Lipid-ASO conjugates were administered systemically or locally in the brain of mice and evaluated for tissue level and cellular level distribution by imaging, qPCR and single-cell RNA sequencing, simultaneously annotating cell origin and global transcriptional changes within the cell. Following systemic administration in mice, lipid conjugation improved overall multi organ efficacy compared to unconjugated ASO, but with pronounced tissue specific differences. Single cell sequencing of liver and heart transcriptomes revealed lipid dependent cellular uptake patterns and transcriptional responses distinct from administration of unconjugated ASO. After intracerebroventricular administration, selected fatty acid conjugates enhanced silencing in deep brain regions such as the striatum, whereas cholesterol conjugation impaired functional delivery despite increased CNS retention. Light-sheet microscopy showed restricted parenchymal penetration of cholesterol ASOs compared with broader but heterogeneous distribution of palmitic acid conjugate. Together, these findings demonstrate that lipid identity critically determines ASO efficacy, productive cellular uptake, and regional CNS engagement, emphasizing the need for context specific lipid design in ASO therapeutic development.

pharmacology and toxicology↗

bioRxiv2026

Click Chemistry-Based Strategy for Modular Ligand Attachment to siRNAs: Toward Extrahepatic RNAi

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b7af11org.highwire.dtl.DTLVardef@146b9c6org.highwire.dtl.DTLVardef@4e8948org.highwire.dtl.DTLVardef@16fa07_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Large-scale expansions and replication stalling of Friedreich's ataxia GAA repeats in an experimental mammalian system

1Human disease Friedreichs ataxia (FRDA) is caused by large-scale expansions of (GAA)n repeats in the first intron of the FXN gene. While repeat expansions during intergenerational transmissions are causative for the disease development, somatic expansions additionally contribute to the disease progression. We and others have previously shown that (GAA)n repeats transiently pause the replication fork progression in cultured human cells. However, whether and by which mechanisms fork stalling underlies repeat expansions remained unclear. Here we developed a new genetically tractable experimental system to simultaneously analyze repeat-mediated fork stalling and large-scale repeat expansions in cultured human cells. It is based on a mammalian/yeast shuttle vector that can transiently replicate from the SV40 replication origin in human HEK-293T cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6; it also contains a cassette for selecting repeat expansions in yeast. Repeat expansions accumulate in mammalian cells and are then detected upon plasmid transformation into yeast. We found that large-scale expansions of (GAA)n repeats do occur in this experimental mammalian system. Further, we observed that repeat expansions frequency depends on several previously implicated proteins in replication fork stalling, reversal, and restart. These proteins include SHPRH, RAD52, ZRANB3, DDX11, SMARCAL1, HLTF, RECQ1 and WRN. Therefore, we propose that GAA repeat expansions might occur as a consequence of deregulated replication fork regression and restoration process.

genetics↗

bioRxiv2022

Large-scale expansions of Friedreich's ataxia GAA·TTC repeats in human cells are prevented by LNA-DNA oligonucleotides and PNA oligomers

The human disease Friedreichs ataxia (FRDA) is caused by expansions of GAA*TTC repeats in the first intron intron of the frataxin (FXN) gene, and both intergenerational and somatic expansions are crucial for disease development. We and others have shown earlier that expanded GAA*TTC repeats can form an intramolecular triplex structure (H-DNA). Here we studied the effects of locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers on the expansion of GAA*TTC repeats in cultured human cells. Our experimental system employes a mammalian/yeast shuttle plasmid containing a selectable cassette to detect repeat expansions. Using our in-house in vitro triplex-specific DNA cleavage assay, we first confirmed H-DNA formation by the (GAA)100*(TTC)100 repeat in the selectable cassette and demonstrated that the designed LNA-DNA oligonucleotides as well as PNA oligomers are able to disrupt this structure. We then found that both LNA-DNA mixmers and PNA oligomers prevent repeat expansions in human cells. In the accompanying paper, we show that expansions of GAA*TTC repeats in this experimental system occur during replication fork stalling, regression and restart at the repetitive run. We hypothesize, therefore, that triplex DNA formation by the GAA*TTC repeats is a key to their instability, while LNA-DNA oligonucleotides and PNA oligomers counteract repeat expansions by disrupting the triplex at the fork or preventing triplex formation upon fork reversal.

genetics↗