Biology subjects

Das, V. R. A.

Publications and source records attributed to Das, V. R. A..

2 recordsLinked to original sources

bioRxiv2025

A Versatile, Simple, and Rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for molecular biology applications

Gene cloning, site-directed mutagenesis (SDM), and short gene synthesis are essential tools in molecular biology, yet existing approaches remain constrained by efficiency, flexibility, and cost. Here, we developed a versatile, simple, and rapid (VSR) 3-in-1 method that integrates seamless cloning, mutagenesis, and short gene assembly in a single platform. This approach works on the principles of overlap extension polymerase chain reaction (OPCR), uses high-fidelity DNA polymerase and short overlapping primers. Target genes can be directly amplified from genomic DNA or complementary DNA (cDNA) templates and cloned into vectors without intermediate purification, restriction enzyme digestion, or ligation. VSR supports the insertion of DNA fragments ranging from 80 bp to 33 kb at any plasmid locus while enabling the introduction of single or multiple nucleotide substitutions at one or more sites in a single reaction. Using this method, we cloned 35 genes of diverse lengths and two complete viral genomes with efficiencies ranging from 60-100% and introduced up to 17 substitutions in a single mutagenesis reaction. We further demonstrate efficient de novo synthesis of short genes from overlapping oligonucleotides. The VSR method is a reliable, rapid, and cost-effective approach with a wide range of applications for both routine and high-throughput workflows. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/682745v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1295a5aorg.highwire.dtl.DTLVardef@1eacb4eorg.highwire.dtl.DTLVardef@1c98fb1org.highwire.dtl.DTLVardef@1f8bcbe_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Green tea and Spirulina extracts inhibit SARS, MERS, and SARS-2 spike pseudotyped virus entry in vitro

Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike (S) protein to bind to a host cell surface receptor. The rapid outbreak of emerging novel coronavirus, SARS-CoV 2 in China inculcates the need for the development of hasty and effective intervention strategies. Medicinal plants and natural compounds have been traditionally used to treat viral infections. Here, we generated VSV based pseudotyped viruses (pvs) of SARS-, MERS-, and SARS-2 CoVs to screen entry inhibitors from natural products. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind on their receptors and enter into the cells. SARS and MERS polyclonal antibodies neutralize SARSpv and SARS-2pv, and MERSpv respectively. Incubation of soluble ACE2 inhibited entry of SARS and SARS-2 pvs but not MERSpv. In addition, expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARSpv, SARS-2pv, and MERSpv respectively. Next, we showed the antiviral properties of known enveloped virus entry inhibitors, Spirulina and Green tea extracts against CoVpvs. SARSpv, MERSpv, and SARS-2pv entry were blocked with higher efficiency when preincubated with either green tea or spirulina extracts. Green tea provided a better inhibitory effect than the spirulina extracts by binding to the S1 domain of spike and blocking the interaction of spike with its receptor. Further studies are required to understand the exact mechanism of viral inhibition. In summary, we demonstrate that pseudotyped virus is an ideal tool for screening viral entry inhibitors. Moreover, spirulina and green tea could be promising antiviral agents against emerging viruses.

microbiology↗