Biology subjects

Chandrasekhar, S.

Publications and source records attributed to Chandrasekhar, S..

8 recordsLinked to original sources

bioRxiv2026

DNA Double-decker Ring Scaffolded Nanodisc for Self-assembly of Membrane Protein into Lipid Bilayer

Membrane models of scaffolded discoidal lipid bilayers called nanodiscs have proven to be a valuable tool for the study of membrane proteins in a native environment. DNA-scaffolded membrane model has emerged as an alternative tool for membrane protein studies. Taking advantage of the designability of DNA nanostructure, we created a double-decker double-stranded DNA ring (DDring) to self-assemble DNA-based nanodiscs (DNA-ND). The DDring is 17 nm wide and 4 nm high, and equipped with 28 alkyl chains on the inside that can interact with each hydrophobic leaflet of the lipid bilayer. We further demonstrate the functionality of DNA-ND membrane model with the assembly of membrane proteins. DDrings are suited to neutral or cationic charged phospholipids and detergents. This study provides more insights into the potential use of DNA- assisted nanodiscs for membrane protein characterization.

bioengineering↗

bioRxiv2026

Peptide-DNA Conjugates for Formation of Lipid Nanodiscs

Nanoscale lipid bilayer mimetics are powerful tools for research on lipid bilayer, membrane proteins or for drug delivery. Established nanoscale bilayer systems that are stabilized by short peptides or polymers produce a broad size distribution and are difficult to customize. Here we introduce a DNA nanotechnology-based lipid bilayer mimetic, in which we covalently conjugated established nanodisc-forming amphiphilic peptides to oligonucleotides. These peptide-DNA conjugates were then hybridized with a circular single-stranded scaffold to form stiff, circular PDC minicircles with 14 peptide modifications at the inner rim of the torus. Lipid reconstitution yielded defined nanodisc with a tightly controlled circumference and component stoichiometry. Molecular dynamics simulations further validated the structural stability and reveal an asymmetric migration of the DNA to one rim of the bilayer. To mimic membrane protein insertion, we co-reconstituted a transmembrane peptide coupled to a bulky quantum dot. In future applications, the size and peptide arrangement can easily be modified in these DNA-templated PDC nanodiscs.

biochemistry↗

bioRxiv2026

Localized heme sensing through a ternary molecular glue

Molecular glues are an emerging class of therapeutics that stabilize binary interactions and there-by rewire disease-relevant protein networks. Whether glues can integrate additional information to orchestrate signaling beyond initial complex formation is unknown. Here, we report that cells use an endogenous glue strategy to sense heme, an essential metabolite with deleterious pro-oxidant properties. Distinct from other glues, heme bridges three polypeptides to trigger degradation of the transcriptional repressor BACH1 through cytoplasmic, but not mitochondrial, CUL2FEM1B. This mechanism allows cells to eliminate toxic heme in the cytoplasm by inducing expression of the heme-degrading oxygenase HMOX1, yet ignore mitochondrial heme destined for function in the electron transport chain. While protective in healthy cells, ternary glue signaling creates a therapeutic vulnerability for Acute Myeloid Leukemias dependent on high rates of ETC assembly. Molecular glues can therefore drive assembly of higher-order complexes to establish localized signaling, which offers unexplored opportunities for induced proximity therapeutics.

biochemistry↗

bioRxiv2026

DNA-Lipid Nanodiscs with a Polyethylene Glycol Interface

Nanoscale bilayer mimetics such as protein or polymer-based nanodiscs are versatile tools to study the physical chemistry of lipid bilayers or the structures and functions of membrane proteins. Here, we introduce DNA-Lipid Nanodiscs (DLNs) in which the interface between hydrophobic lipids and the charged DNA is mediated through amphiphilic poly(ethylene)glycol (PEG). For this, we modified oligonucleotides with PEG and hybridized them to a single-stranded ring to form functionalized minicircles with a well-defined diameter. The center of these minicircles can be filled with a lipid bilayer through addition of detergent-solubilized lipids followed by detergent removal. Simulations reveal that the methylene groups in PEG form dynamic interactions with the acyl chains of lipids, effectively shielding the hydrophobic mismatch. As proof of concept towards incorporation of complex membrane proteins, we inserted the biotinylated transmembrane domain of synaptobrevin into these nanodiscs and bound them to streptavidin-modified quantum dots as a marker for successful incorporation. We envision these atomically precise, modular DNA scaffolds to be widely applicable in future studies of membrane proteins and nanoscale lipid membranes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/705827v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@17cfcb5org.highwire.dtl.DTLVardef@b2dd2corg.highwire.dtl.DTLVardef@d6899aorg.highwire.dtl.DTLVardef@e400c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

S-Alkyl-Phosphorothioate Modifications Reduce Thermal and Structural Stability of DNA Duplexes

While phosphorothioate (PS) oligonucleotides are usually used in therapeutic applications, they also offer the cheapest and synthetically most straightforward route to introduce hydrophobic modifications for applications in structural DNA nanotechnology and biophysics. For this, the sulfur atom is S-alkylated with alkyl iodides, enabling a hydrophobically tunable interface of DNA nanostructures with lipid bilayers. While longer and more alkyls per helical turn should lead to stronger interactions with lipid membranes, we found that excessive S-alkylations strongly inhibit hybridization of oligonucleotides to their complementary strands and decrease their melting temperature, despite a reduction in electrostatic repulsion between the two strands. Moreover, both the type and placement of alkyl modifications influence the melting temperature. Atomistic molecular dynamics simulations reveal two complementary mechanisms that explain the experimental findings. First, S-alkylated oligonucleotides are more compact and less dynamic than unmodified ones, likely inhibiting their ability to hybridize to their complementary strands. Second, S-alkyls in double-stranded DNA promote defect formation due to alkyl modifications having hydrophobic interactions with other alkyl groups and nucleobases, therefore reducing the thermal and structural stability of alkylated DNA duplexes. This study serves as a practical guide for tuning hydrophobicity while maintaining structural stability in membrane-interfacing DNA nanostructures.

biochemistry↗

bioRxiv2024

Bending Unwinds DNA

DNA is under high bending and torsional strain in many biological contexts 1,2, but elastic limits of tightly bent DNA under simultaneous torsional strain are not fully understood 3-22. We synthesized DNA circles with all possible radii of curvature between r {approx} 2.7 - 5.7 nm in increments of{Delta} r = 0.05 nm and defined helical repeats ranging from h {approx} 10 - 13 base pairs per helical turn. Nuclease digest reveals that DNA can be bent to r {approx} 3.0 nm without kinking, but only when h < ~10.9 bp/turn, while underwound DNA kinks irrespective of curvature. Histone proteins overwind DNA and thereby mechanically stabilize it. The natural helical repeat h0 increases from 10.45 to >11 bp/turn due to twist-bend coupling, which is not caused by kinking before ligation. These findings require reassessing our models and the energetics of molecular mechanisms involving DNA under mechanical stress.

biophysics↗

bioRxiv2022

AGO2 silences mobile transposons in the nucleus of quiescent cells

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells where mTOR signaling is low, AGO2 accumulates in the nucleus where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

molecular biology↗

bioRxiv2021

Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within [~]1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

microbiology↗