Biology subjects

Vangaveti, S.

Publications and source records attributed to Vangaveti, S..

9 recordsLinked to original sources

bioRxiv2026

Multi-dimensional DNA nanostructures isothermally assembled in hydrated ionic liquids

DNA nanostructures can be tailored to perform a wide variety of functions, with continued interest in biological applications. Some aspects of DNA nanostructure assembly can hinder the ability of nanostructures to be useful in physiological environments. Typical assembly methods employ magnesium ions to stabilize the structure, which leave the structure susceptible to damage by nucleases in body fluids. Further, DNA nanostructure assembly typically involves a thermal annealing protocol in which DNA strands are heated in a specific buffer to a high temperature and cooled slowly at specific rates, preventing convenient encapsulation of temperature-sensitive guest molecules. In this work, we demonstrate the assembly of a wide variety of DNA nanostructures and 3D crystals in a hydrated ionic liquid (choline dihydrogen phosphate, CDHP) instead of magnesium at constant moderate temperatures, thus avoiding thermal annealing. CDHP-assembled structures show enhanced biostability against a variety of nucleases. Molecular dynamics simulations show that choline ions stabilize DNA nanostructures by a direct and close-range interaction in contrast to the predominantly water-mediated interactions of Mg2+, leading to enhanced nuclease resistance in CDHP-containing environments. CDHP-assembled structures do not affect the viability of HepG2 cells and show higher cell internalization. Overall, this work develops a potential method to construct more biostable DNA nanostructures and 3D crystals in a simple one pot process. Assembly of DNA nanostructures under isothermal conditions is desirable for scaffolding biomolecules and to reduce the need for thermal annealing instruments, allowing nanostructure preparation in low-resource settings.

biophysics↗

bioRxiv2025

Investigating polarity effects in DNA base stacking

Nucleic acid structures are stabilized by both base pairing and base stacking. While energetics of base pairing interactions are relatively well established, our understanding of the energetic contributions of base stacking remain incomplete. Here, we use a combination of single-molecule and computational biophysics approaches to investigate the effect of strand polarity on base-stacking energetics. We designed pairs of DNA constructs with reversed stacking polarities at nick sites, along with corresponding no-stack controls to isolate stacking contributions. Performing single-molecule force-clamp assays with a Centrifuge Force Microscope (CFM), we observed polarity-dependent differences in stacking energetics. These differences were most pronounced in purine-purine and certain purine-pyrimidine interactions. Notably, a 5' purine stacked on a 3' pyrimidine was generally more stable than the reverse polarity. We employed molecular dynamics (MD) simulations to observe stacking interfaces in the DNA constructs. The simulations were qualitatively consistent with our experiments, and showed positional differences between opposite polarity stacking pairs, giving some insight into the origin of these polarity differences. Overall, these results demonstrate that base polarity can modulate stacking stability and should be considered when designing short duplex regions such as overhangs in molecular biology and biotechnology applications.

biophysics↗

bioRxiv2025

Temporal Control of CRISPR-Cas9 Activity Using Bio-orthogonal Chemistry.

The CRISPR-Cas9 system has become a widely used tool for genome engineering. Here we present a new method for small-molecule control of CRISPR-Cas9 using bio-orthogonal chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). We carried out molecular modeling studies and identified a unique position on single guide RNA (sgRNA) that can be site-specifically tagged with Tz without disrupting its activity. We also synthesized a series of TCO-modified CRISPR suppressors. When exogenously added, they click to the Tz-tagged sgRNA, perturb the system and drastically reduce the nuclease activity. The most successful suppressor is a TCO-modified six amino acid long cell-penetrating peptide, which shows excellent cell permeability. We showed that out method to control CRISPR-Cas9 nuclease activity is general by applying it to three different sgRNAs. We also showed that our method works in solution, as well as live HEL293 cells. We utilized flow cytometry to demonstrate temporal control of CRISPR-Cas9 targeting GFP. Lastly, we showed the therapeutic potential of our method by targeting vascular endothelial growth factor A (VEGFA).

biochemistry↗

bioRxiv2024

Influence of metal ions on the isothermal self-assembly of DNA nanostructures

DNA nanostructures are typically assembled by thermal annealing in buffers containing magnesium. We demonstrate the assembly of DNA nanostructures at constant temperatures ranging from 4 {degrees}C to 50 {degrees}C in solutions containing different metal ions. The choice of metal ions and the assembly temperature influence the isothermal assembly of several DNA motifs and designed three-dimensional DNA crystals. Molecular dynamics simulations show more fluctuations of the DNA structure in select monovalent ions (Na+ and K+) compared to divalent ions (Mg2+ and Ca2+). A key highlight is the successful assembly of DNA motifs in nickel-containing buffer at temperatures below 40 {degrees}C, otherwise unachievable at higher temperatures, or using an annealing protocol. DNA nanostructures isothermally assembled in different ions do not affect the viability of fibroblasts, myoblasts, and myotubes and or the immune response in myoblasts. The use of ions other than the typically-used magnesium holds key potential in biological and materials science applications that require minimal amounts of magnesium.

bioengineering↗

bioRxiv2024

CAG repeat-selective compounds reduce abundance of expanded CAG RNAs in patient cell and murine models of SCAs

Spinocerebellar ataxias (SCAs) are a genetically heterogenous group of devastating neurodegenerative conditions for which clinical care currently focuses on managing symptoms. Across these diseases there is an unmet need for therapies that address underlying disease mechanisms. We utilised the shared CAG repeat expansion mutation causative for a large subgroup of SCAs, to develop a novel disease-gene independent and mechanism agnostic small molecule screening approach to identify compounds with therapeutic potential across multiple SCAs. Using this approach, we identified the FDA approved microtubule inhibitor Colchicine and a novel CAG-repeat binding compound that reduce expression of disease associated transcripts across SCA1, 3 and 7 patient derived fibroblast lines and the Atxn1154Q/2Q SCA1 mouse model in a repeat selective manner. Furthermore, our lead candidate rescues dysregulated alternative splicing in Atxn1154Q/2Q mice. This work provides the first example of small molecules capable of targeting the underlying mechanism of disease across multiple CAG SCAs.

molecular biology↗

bioRxiv2024

Elucidating the influence of RNA modifications and Magnesium ions on tRNAPhe conformational dynamics in S. cerevisiae: Insights from Replica Exchange Molecular Dynamics simulations

Post-transcriptional modifications in RNA can significantly impact their structure and function. In particular, transfer RNAs (tRNAs) are heavily modified, with around 100 different naturally occurring nucleotide modifications contributing to codon bias and decoding efficiency. Here, we describe our efforts to investigate the impact of RNA modifications on the structure and stability of tRNA Phenylalanine (tRNAPhe) from S. cerevisiae using molecular dynamics (MD) simulations. Through temperature replica exchange MD (T-REMD) studies, we explored the unfolding pathway to understand how RNA modifications influence the conformational dynamics of tRNAPhe, both in the presence and absence of magnesium ions (Mg2+). We observe that modified nucleotides in key regions of the tRNA establish a complex network of hydrogen bonds and stacking interactions which is essential for tertiary structure stability of the tRNA. Furthermore, our simulations show that modifications facilitate the formation of ion binding sites on the tRNA. However, high concentrations of Mg2+ ions can stabilize the tRNA tertiary structure in the absence of modifications. Our findings illuminate the intricate interactions between modifications, magnesium ions, and RNA structural stability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/584441v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@a8d8e1org.highwire.dtl.DTLVardef@136a038org.highwire.dtl.DTLVardef@150bf34org.highwire.dtl.DTLVardef@391279_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Mutation of two intronic nucleotide alters RNA structure and dynamics, inhibiting MBNL1 and RBFOX1 regulated splicing of the Insulin Receptor

Alternative splicing (AS) of Exon 11 of the Insulin Receptor (INSR) is highly regulated and disrupted in several human disorders. To better understand INSR exon 11 AS regulation, splicing activity of an INSR exon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/574689v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e410org.highwire.dtl.DTLVardef@7deb00org.highwire.dtl.DTLVardef@cf69f9org.highwire.dtl.DTLVardef@13684e8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure:C_FLOATNO Model for INSR exon 11 splicing regulation through the UGCAUG motif. We propose that the UGCAUG motif, MBNL1, and RBFOX1 cooperatively regulate INSR exon 11 splicing. Mutating this UGCAUG motif is sufficient to alter RNA structural dynamics to disrupt this regulation. C_FIG

biochemistry↗

bioRxiv2023

Unusual Structural Properties of Switchback DNA

Synthetic DNA motifs form the basis of nucleic acid nanotechnology, and their biochemical and biophysical properties determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

bioengineering↗

bioRxiv2023

Quercetin selectively reduces expanded repeat RNA levels in models of myotonic dystrophy

Myotonic dystrophy is a multisystemic neuromuscular disease caused by either a CTG repeat expansion in DMPK (DM1) or a CCTG repeat expansion in CNBP (DM2). Transcription of the expanded alleles produces toxic gain-of-function RNA that sequester the MBNL family of alternative splicing regulators into ribonuclear foci, leading to pathogenic mis-splicing. There are currently no approved treatments that target the root cause of disease which is the production of the toxic expansion RNA molecules. In this study, using our previously established HeLa DM1 repeat selective screening platform, we identified the natural product quercetin as a selective modulator of toxic RNA levels. Quercetin treatment selectively reduced toxic RNA levels and rescued MBNL dependent mis-splicing in DM1 and DM2 patient derived cell lines and in the HSALR transgenic DM1 mouse model where rescue of myotonia was also observed. Based on our data and its safety profile for use in humans, we have identified quercetin as a priority disease-targeting therapeutic lead for clinical evaluation for the treatment of DM1 and DM2. One Sentence SummaryThe natural product quercetin reduces toxic RNA in myotonic dystrophy.

molecular biology↗