Biology subjects

Kohli, R.

Publications and source records attributed to Kohli, R..

6 recordsLinked to original sources

bioRxiv2026

Genetic Code Expansion for Site-Specific Encoding of a Switchable, Intrinsic Fluorophore-Quencher Pair to Monitor Protein Dynamics

Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore-quencher pair comprising two non-canonical amino acids--acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet) -- into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by biorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern-Volmer quenching, fluorescence lifetime measurements, and "proline ruler" peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: 1) calmodulin, a calcium-sensing protein, 2) RecA, a DNA damage sensor in bacteria, and 3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, enables high-throughput drug screening, and advances tools for studying protein structure-function relationships.

biochemistry↗

bioRxiv2024

Critical assessment of nanopore sequencing for the detection of multiple forms of DNA modifications

While nanopore sequencing is increasingly used for mapping DNA modifications, it is important to recognize associated false-positive calls, as they can mislead biological interpretations. To assist biologists and methods developers, we describe a framework, modFDR, for rigorous evaluation that emphasizes the use of the false discovery rate with rationally designed negative controls capturing both general background and confounding modifications. Our critical assessment across multiple DNA modifications shows that while nanopore sequencing performs reliably for high-abundance modifications--including 5-methylcytosine (5mC) at CpG sites in mammalian cells and 5-hydroxymethylcytosine (5hmC) in mammalian brain cells--it produces a substantial fraction of false-positive detections for low-abundance modifications, such as 5mC at CpH sites, 5hmC, and N6-methyldeoxyadenine (6mA) in most mammalian cell types. Although newer models improve certain aspects, systematic false positives remain, and we further observe elevated false negatives for 5mCpG when benchmarked against orthogonal enzymatic methods. This study highlights the urgent need to incorporate modFDR into future methods development, evaluation, and biological studies, and advocates prioritizing nanopore sequencing for mapping abundant rather than rare modifications in biomedical applications.

genomics↗

bioRxiv2024

Long somatic DNA-repeat expansion drives neurodegeneration in Huntington disease

Huntington Disease (HD) is a fatal genetic disease in which most striatal projection neurons (SPNs) degenerate. The central biological question about HD pathogenesis has been how the disease-causing DNA repeat expansion (CAGn) in the huntingtin (HTT) gene leads to neurodegeneration after decades of apparent latency. Inherited HTT alleles with a longer CAG repeat hasten disease onset; the length of this repeat also changes over time, generating somatic mosaicism, and genes that regulate DNA-repeat stability can influence HD age-at-onset. To understand the relationship between a cells CAG-repeat length and its biological state, we developed a single-cell method for measuring CAG-repeat length together with genome-wide RNA expression. We found that the HTT CAG repeat expands from 40-45 CAGs to 100-500+ CAGs in HD-vulnerable SPNs but not in other striatal cell types, with these long DNA-repeat expansions acquired at different times by individual SPNs. Surprisingly, somatic expansion from 40 to 150 CAGs had no apparent effect upon gene expression - but neurons with 150-500+ CAGs shared profound gene-expression changes. These expression changes involved hundreds of genes, escalated alongside further CAG-repeat expansion, eroded positive and then negative features of neuronal identity, and culminated in expression of senescence/apoptosis genes. Rates of striatal neuron loss across HD stages reflected the rates at which neurons entered this biologically distorted state. Our results suggest that HTT CAG repeats in striatal neurons undergo decades of biologically quiet expansion, then, as they asynchronously cross a high threshold, cause SPNs to degenerate quickly and asynchronously. We conclude that, at any moment in the course of HD, most neurons have an innocuous (but unstable) huntingtin gene, and that HD pathogenesis is a DNA process for almost all of a neurons life.

genetics↗

bioRxiv2023

Structure and symmetries of the genetic codes for vertebrate, ascidian and yeast mitochondria

Genetic codes assign sixty-four codons to twenty amino acids. These assignments are known to follow certain rules. One question long considered but still unresolved is if these rules are derived from an underlying structure in genetic codes. Knowledge of such structure could facilitate better understanding of the biochemical, physico-chemical and evolutionary causes of the observed codon assignments. Our first finding reveals a coherent and symmetric structure in the genetic codes of vertebrate, ascidian, and yeast mitochondria. This structure is derived from a "simple" code that assigns all four codons with the same second nucleotide to a single amino acid if the first nucleotide is C or G, and assigns pairs of codons with the same second nucleotide to a single amino acid if the first nucleotide is A or U, and their third nucleotides are both purines (A and G) or both pyrimidines (U and C). The translation mechanism for the three mitochondria reflects this structure, one tRNA decoding each group of two or four codons into an amino acid. Our second finding is that the mycoplasma/spiroplasma and standard genetic codes are obtained by small sequential modifications of the vertebrate mitochondrial code and retain almost all its symmetries. We use group theory to characterize the symmetries of the simple and mitochondrial codes, and speculate on the implications of the structure for detecting translation errors and the evolution of the genetic code.

genetics↗

bioRxiv2022

Interactive Neurorobotics: Behavioral and Neural Dynamics of Agent Interactions

Interactive neurorobotics is a subfield which characterizes brain responses evoked during interaction with a robot, and their relationship with the behavioral responses. Gathering rich neural and behavioral data from humans or animals responding to agents can act as a scaffold for the design process of future social robots. The goals of this research can be broadly broken down into two categories. The first, seeks to directly study how organisms respond to artificial agents in contrast to biological or inanimate ones. The second, uses the novel affordances of the robotic platforms to investigate complex phenomena, such as responses to multisensory stimuli during minimally structured interactions, that would be difficult to capture with classical experimental setups. Here we argue that to realize the full potential of the approach, both goals must be integrated through methodological design that is informed by a deep understanding of the model system, as well as engineering and analytical considerations. We then propose a general framework for such experiments that emphasizes naturalistic interactions combined with multimodal observations and complementary analysis pipelines that are necessary to render a holistic picture of the data for the purpose of informing robotic design principles. Finally, we demonstrate this approach with an exemplar rat-robot social interaction task which included simultaneous multi-agent tracking and neural recordings.

animal behavior and cognition↗

bioRxiv2021

Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation. ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

microbiology↗