Biology subjects

Gavrilov, M.

Publications and source records attributed to Gavrilov, M..

4 recordsLinked to original sources

bioRxiv2024

Reliable amplification of highly repetitive or low complexity sequence DNA enabled by superhelicase-mediated isothermal amplification

PCR is a cornerstone of molecular biology, but many biologically important DNA templates remain difficult to amplify. Long tandem repeats, low-complexity tracts, and sequences with extreme base composition often yield low product levels, smeared bands, stutter products, or truncated amplicons. These failures can arise because repeated cycles of thermal denaturation and reannealing promote off-register annealing, polymerase slippage, secondary-structure formation, and incomplete extension. Previously, we developed SSB-Helicase Assisted Rapid PCR (SHARP), an isothermal amplification method in which an engineered superhelicase and single-stranded DNA-binding protein replace the thermal melting step of PCR with enzymatic strand separation. Here, we tested whether SHARP can improve amplification of templates that are refractory to conventional PCR. SHARP robustly amplified up to six identical tandem repeats of the Widom 601 nucleosome-positioning sequence and up to 35 identical ankyrin repeats, targets that were poorly amplified by conventional PCR under the conditions tested. SHARP also amplified templates with extreme base composition, including a 95% AT-rich template and GC-rich templates (up to ~95% GC), as well as a 99-repeat CGG tract associated with Fragile X syndrome and a CAG/CAA repeat tract from mutant HTT exon 1 associated with Huntington disease. Together, these results show that helicase-driven isothermal amplification can expand access to repetitive and compositionally extreme DNA sequences that are challenging for conventional thermocycling-based PCR.

bioengineering↗

bioRxiv2023

A novel framework to build saliva-based DNA methylation biomarkers: quantifying systemic chronic inflammation as a case study

Accessible and non-invasive biomarkers that measure human ageing processes and the risk of developing age-related disease are paramount in preventative healthcare. In this study, we describe a novel framework to train saliva-based DNA methylation (DNAm) biomarkers that are reproducible and biologically interpretable. By leveraging a reliability dataset with replicates across tissues, we demonstrate that it is possible to transfer knowledge from blood DNAm data to saliva DNAm data using DNAm proxies of blood proteins (EpiScores). We then apply these methods to create a new saliva-based epigenetic clock (InflammAge) that quantifies systemic chronic inflammation (SCI) in humans. Using a large blood DNAm human cohort with linked electronic health records and over 18,000 individuals (Generation Scotland), we demonstrate that InflammAge significantly associates with all-cause mortality, disease outcomes, lifestyle factors and immunosenescence; in many cases outperforming the widely used SCI biomarker C-reactive protein (CRP). We propose that our biomarker discovery framework and InflammAge will be useful to improve our understanding of the molecular mechanisms underpinning human ageing and to assess the impact of gero-protective interventions.

bioinformatics↗

bioRxiv2022

Achieving single nucleotide sensitivity in direct hybridization genome imaging

Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, sgGOLDFISH, which leverages the high cleavage specificity of enhanced Cas9 combined with a single extended guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. Using sgGOLDFISH, we identified base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validated the identification through progerin immunofluorescence, and demonstrated accurate sub-nuclear localization of point mutations.

genomics↗

bioRxiv2022

Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution.

We used single-molecule nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of Superfamily 1 helicase PcrA as it translocates on, or unwinds, several kb-long DNA molecules. We recorded over 2 million enzyme steps under various assisting and opposing forces in diverse ATP and ADP conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo, such as when the helicase encounters bound proteins or duplex DNA; we show how PcrAs kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base-pairs being unwound. Instead, the sequence of the single stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

biophysics↗