Biology subjects

Sengar, A.

Publications and source records attributed to Sengar, A..

6 recordsLinked to original sources

bioRxiv2023

Kinetic Proofreading can Enhance Single Nucleotide Discrimination in a Non-enzymatic DNA Strand Displacement Network

Kinetic proofreading is used throughout natural systems to enhance the specificity of molecular recognition. At its most basic level, kinetic proofreading uses a supply of chemical fuel to drive a recognition interaction out of equilibrium, allowing a single free-energy difference between correct and incorrect targets to be exploited two or more times. Despite its importance in biology, there has been little effort to incorporate kinetic proofreading into synthetic systems in which molecular recognition is important, such as nucleic acid nanotechnology. In this article, we introduce a DNA strand displacement-based kinetic proofreading motif, showing that the consumption of a DNA-based fuel can be used to enhance molecular recognition during a templated dimerization reaction. We then show that kinetic proofreading can enhance the specificity with which a probe discriminates single nucleotide mutations, both in terms of the initial rate with which the probe reacts and the long-time behaviour.

biophysics↗

bioRxiv2023

Overcoming the speed limit of four-way DNA branch migration with bulges in toeholds

Dynamic DNA nanotechnology involves the use of DNA strands to create programmable reaction networks and nanodevices. The key reaction in dynamic DNA nanotechnology is the exchange of DNA strands between different molecular species, which is achieved through three-way and four-way DNA exchange reactions. While both of these reactions have been widely used to build reaction circuits, the four-way exchange reaction has traditionally been slower and less efficient than the three-way reaction. In this paper, we describe a new mechanism to optimise the kinetics of the four-way DNA exchange reaction by adding bulges to the toeholds of the four-way DNA complexes involved in the reaction. These bulges facilitate an alternative branch migration mechanism and destabilise the four-way DNA junction, increasing the branch migration rate and unbinding rate of the four-way exchange reaction, bringing it closer to the kinetics of the three-way reaction. This new mechanism has the potential to expand the field of dynamic DNA nanotechnology by enabling efficient four-way DNA exchange reactions for in vivo applications.

biophysics↗

bioRxiv2022

The ACE-2 receptor accelerates but is not biochemically required for SARS-CoV-2 membrane fusion.

The SARS-CoV-2 coronavirus infects human cells via the ACE-2 receptor. Circumstantial evidence suggests that ACE-2 may not just serve as an attachment factor but also help activate the SARS-CoV-2 spike protein for membrane fusion. Here, we test that hypothesis directly, using DNA-lipid tethering as a synthetic attachment factor in the place of ACE-2. We find that SARS-CoV-2 pseudovirus and viruslike particles are both capable of membrane fusion if attached in the absence of ACE-2 and activated with an appropriate protease. However, addition of soluble ACE-2 speeds the fusion reaction. This is observed for both the Wuhan strain and the B.1.1.529 Omicron variant. Kinetic analysis suggests that there are at least two rate-limiting steps for SARS-CoV-2 membrane fusion, one of which is ACE-2 dependent and one of which is not. These data establish that, in the presence of an alternative attachment factor, ACE-2 is not biochemically required for SARS-CoV-2 membrane fusion. Since ACE-2 serves as the high-affinity attachment factor on human cells, the possibility to replace it with other factors has implications for the evolvability of SARS-CoV-2 and the fitness landscape for future related coronaviruses.

microbiology↗

bioRxiv2022

Mechanistic dissection of antibody inhibition of influenza entry yields unexpected heterogeneity

Neutralizing antibodies against influenza have generally been classified according to their recognition sites, with antibodies against the head domain of hemagglutinin thought to inhibit attachment and antibodies against the stalk region thought to inhibit fusion. Here, we report the development of a microfluidic assay to measure neutralization of viral entry that can clearly differentiate between effects on attachment and fusion. Testing multiple broadly-neutralizing antibodies against the hemagglutinin stalk domain, we obtain a surprising result: some broadly-neutralizing antibodies inhibit fusion only, while others inhibit both fusion and viral attachment. Antibodies binding the globular head domain primarily inhibit attachment but can also reduce the fusogenic capability of viral particles that nonetheless bind receptor. These findings shed light on the unexpectedly heterogeneous mechanisms of antibody neutralization even within similar recognition sites. The assay we have developed also provides a tool to optimize vaccine design by permitting assessment of the elicited antibody response with greater mechanistic resolution.

biophysics↗

bioRxiv2021

Single-virus fusion measurements yield an opportunistic model for SARS-CoV-2 fusion

SARS-CoV-2 binds to cell-surface receptors and is activated for membrane fusion and cell entry via proteolytic cleavage. Phenomenological data have shown that SARS-CoV-2 can be activated for entry at either the cell surface or in endosomes, but the relative roles in different cell types and mechanisms of entry have been debated. Here we use single-virus fusion experiments and exogenously controlled proteases to probe activation directly. We find that plasma membrane and an appropriate protease are sufficient to support SARS-CoV-2 pseudovirus fusion. Furthermore, fusion kinetics of SARS-CoV-2 pseudoviruses are indistinguishable no matter which of a broad range of proteases was used to activate the virus. This suggests that fusion mechanism is insensitive to protease identity or even whether activation occurs before or after receptor binding. These data support a model for opportunistic fusion by SARS-CoV-2, where subcellular location of entry likely depends on the differential activity of airway, cell-surface, and endosomal proteases, but all support infection. Inhibiting any single host protease may thus reduce infection in some cells but may be less clinically robust. ImportanceSARS-CoV-2 can use multiple pathways to infect cells, as demonstrated recently when new viral variants switched dominant infection pathways. Here, we use single-virus fusion experiments together with biochemical reconstitution to show that these multiple pathways coexist simultaneously and specifically that the virus can be activated by different proteases in different cellular compartments with mechanistically identical effect. The consequences of this are that the virus is evolutionarily plastic and that therapies targeting viral entry should address multiple pathways at once to achieve optimal clinical effects.

microbiology↗

bioRxiv2020

Alternative splicing of GluN1 gates glycine-primed internalization of NMDA receptors

N-methyl-D-aspartate receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Gating of the NMDARs requires binding of four co-agonist molecules, but the receptors can signal non-ionotropically through binding of glycine, alone, to its cognate site on GluN1. A consequence of this signalling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit is not a singular molecular species in the CNS, rather there are 8 alternatively spliced isoforms of this subunit produced by including or excluding the N1 and the C1, C2 or C2 polypeptide cassettes. Whether alternative splicing affects glycine priming signalling is unknown. Here, using recombinant NMDARs expressed heterologously we discovered that glycine priming of NMDARs critically depends on alternative splicing: the four splice isoforms lacking the N1 cassette, encoded in exon 5, are primed by glycine whereas glycine priming is blocked in the four splice variants containing the N1 cassette. On the other hand, the C-terminal cassettes - C1, C2 or C2 - had no effect on glycine priming signalling. Nor was glycine priming affected by the GluN2 subunit in the receptor. In wild-type mice we found that glycine primed internalization of synaptic NMDARs in CA1 hippocampal pyramidal neurons. With mice we engineered such that GluN1 obligatorily contained the N1 cassette, glycine did not prime synaptic NMDARs in pyramidal neurons. In contrast to pyramidal neurons, we discovered that in wild-type mice, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling non-ionotropic signalling of NMDAR by glycine and the consequential cell surface dynamics of the receptors.

neuroscience↗