Biology subjects

Damha, M. J.

Publications and source records attributed to Damha, M. J..

6 recordsLinked to original sources

bioRxiv2026

Chemical control of 2'-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

Advanced CRISPR-based therapies benefit from CRISPR RNA (crRNA) with high nuclease resistance and enhanced drug-like properties, which is primarily achieved through chemical replacement of the RNA ribose moiety. However, for gene editing enzymes like CRISPR-Cas9 a handful of residues cannot be replaced with chemical ribose analogues, limiting the scope of therapeutic strategies. The mechanism underlying this restriction has remained unclear. Here, using nucleic acid chemistry, biochemistry, cryo-EM, and molecular dynamics simulations, we show that the ribose 2-hydroxyl group at specific crRNA residues is required to achieve a conformational state competent for Cas9 target DNA binding. Based on the mechanistic principles uncovered, we combined site-specific phosphorothioate linkage chemistry with ribose replacement chemistry to restore binding and activity, resulting in high Cas9 editing efficiency and fidelity with a ribose-free crRNA. This study offers novel mechanistic insight and crRNAs with full chemical stabilization, making rational design of guide RNAs with complete nuclease protection for CRISPR-based medicines possible.

biochemistry↗

bioRxiv2025

A guide RNA repeat checkpoint steers CRISPR-Cas9 catalysis

A widely adopted CRISPR-Cas9 modification is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). However, mechanistic and functional differences between dgRNA and sgRNA have not been systematically explored. By investigating the activity of these two guide architectures, we discover a guide RNA repeat checkpoint (GRC) that senses the structure and dynamics of the guide repeat region. The GRC coordinates with other checkpoint mechanisms known to recognize spacer-target R-loop fidelity, together licensing target cleavage. Based on these principles, dgRNA and sgRNA properties could be combined into guide repeat-truncated sgRNAs (grtRNAs) and paired with high-fidelity Cas9 variants to further reduce off target editing. A mechanism that helps govern Cas9 catalysis via guide RNA structure sensing and communicates with other checkpoints offers a previously unappreciated path for understanding and further improving gene editing outcomes.

biochemistry↗

bioRxiv2025

An HIV-1 Reference Epitranscriptome

Post-transcriptional modifications to RNA, which comprise the epitranscriptome, play important roles in RNA metabolism, gene regulation, and human disease, including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported and investigated in the context of virus and host biology. However, the diversity of experimental approaches used has made clear correlations across studies, as well as the significance of the HIV-1 epitranscriptome in biology and disease, difficult to assess. Therefore, we established a reference HIV-1 epitranscriptome. We sequenced the model NL4-3 HIV-1 genome from infected primary CD4+ T cells and the Jurkat cell line using the latest nanopore chemistry, optimized RNA preparation methods, and the most current and readily available base-calling algorithms. A highly reproducible sense and a preliminary antisense HIV-1 epitranscriptome were created, where N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine (psi), inosine, and 2-O-methyl (Nm) modifications could be identified by rapid multiplexed base-calling. We observed that sequence and neighboring modification contexts induced modification miscalling, which could be corrected with synthetic HIV-1 RNA fragments. We validated m6A modification sites with STM2457, a small molecule inhibitor of methyltransferase-like 3 (METTL3). We find that modifications are quite stable under combination antiretroviral therapy (cART) treatment, in primary CD4+ T cells, and in HIV-1 virions. Sequencing samples from people living with HIV (PLWH) revealed conservation of m6A modifications. However, analysis of spliced transcript variants suggests transcript-dependent modification levels. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor, reproducibility, and uniformity across HIV-1 epitranscriptomics studies. They also provide a roadmap for the creation of reference epitranscriptomes for many other viruses or pathogens.

genetics↗

bioRxiv2023

DNA i-motif levels are overwhelmingly depleted in living human cells: insights from in-cell NMR.

I-Motifs (iM) are non-canonical DNA structures potentially forming in accessible, single-stranded, cytosine-rich genomic regions, with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitored iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37{degrees}C. Our findings show that iMFPS displaying pHT<7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT >7 occur as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) revealed that cell cycle-dependent iM formation has a dual origin and iM formation concerns only a small fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

biophysics↗

bioRxiv2020

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

biochemistry↗

bioRxiv2017

A conserved homo-dimerization interface in human IFIT1 provides insights into IFIT interactome assembly

The Interferon-Induced Proteins with Tetratricopeptide Repeats (IFITs) are a group of potently expressed Interferon Stimulated Genes that mediate antiviral innate immunity. Previous studies have revealed that most IFITs partake in higher order structures, potentially as part of an IFIT interactome that results in viral inhibition. Recent crystal structures of a mutated, monomeric form of IFIT1 revealed the molecular basis of how it recognizes non-self, capped viral mRNAs to selectively inhibit their translation. However, wild-type IFIT1 forms dimers in solution and the role of dimerization was not examined in detail. Here we present a structural and biochemical analysis of wild-type IFIT1 in complex with capped and uncapped RNA. Wild-type IFIT1 forms an antiparallel, elongated dimer that is in stark contrast to the domain-swapped, parallel dimer found in IFIT2. Dimerization takes place through a small, C-terminal interface that is evolutionarily conserved in IFIT1 and IFIT1B proteins. The interface is modular and can be grafted onto IFIT5, which is natively monomeric, to induce dimerization. Mutational analysis of this interface showed that homo-dimerization is not required for full RNA binding or translational inhibition by IFIT1. Sedimentation velocity analytical ultracentrifugation measurements demonstrated a reversible monomer-dimer equilibrium, suggesting that dimerization is of low affinity and could play a role under physiological concentrations, possibly in regulating IFIT interactome assembly. Finally, conformational changes in IFIT1 that occur upon RNA binding provide insight into how RNA enters its binding site in solution.

biochemistry↗