The general principles in physics and chemistry stem from the quantitative understanding of the elementary units like atoms. Biology has been beyond such clarity mostly due to our inability to conduct precise quantitative measurements that provide absolute values of various parameters in the cell. We address that limitation by making precise measurements of the key parameters necessary to define a basic cellular system. Using highly sensitive detection of single biological macromolecules like proteins or nucleic acids by both optical and non-optical methods, we determine the molecular behaviour at the single molecule level as well as its distribution at the population level.

SINGLE MOLECULE RNA POLYMERASE DYNAMICS IN LIVING CELLS                                                

Mechanistic understanding of the viral replication process by RNA dependent RNA Polymerases (RdRPs) inside the cells is complicated as it is inherently stochastic. It initiates from a small number of single stranded (ss)RNA molecules (sometimes just one) during its initial phase (i.e. during negative strand synthesis). Complexity further increases as RdRP compartmentalizes in membrane-bound heterogeneous structures for positive strand synthesis from double stranded RNA. Furthermore, RdRP regulation arises from phosphorylation, structural changes or transient interactions with other viral/host proteins that generates a heterogeneous population of replication complexes.It is unclear how such diverse phases of replication are regulated to ensure efficient replication during the infection cycle. To address these issues, we are developing  spectroscopic and imaging tools to study inter-molecular and intra-molecular dynamics in living cells while maintaining single molecule sensitivity.

Related Articles

Roy 2008: A basic review of single molecule FRET technology

Gebhardt 2013: Demonstrates Reflected Light Sheet Microscopy for single molecule imaging in live cells

SUPER-RESOLUTION IMAGING OF CELLULAR ARCHITECTURE                                                           

Nuclear processes like transcription are omnipresent in the nucleus, and their organization beyond the conventional diffraction limited optical microscopy. Using highly precise localization of stochastically blinking fluorescent tags on biomolecules (eg. antibodies or proteins), reconstructed images far beyond (up to 10-20 fold) the optical resolution (which is limited to 200-300 nm) are possible. Moreover, the well understood photophysical properties of current dyes allow us to estimate the average number of molecules at each position in our super-resolution images. The goal of this project will be two fold: a) to address the heterogeneity in the distribution of molecular components involved in gene expression and regulation and b) to quantify the number of molecules involved in macromolecular assemblies that carry out such processes.

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Huang 2009: Review of Super-Resolution Microscopy

Hell 2007: Review of Super-Resolution Microscopy

VIRAL SELF ASSEMBLY                                                                                                                         

Virus particle generation is a classical example of self assembly which is amenable to targeting by drugs. However, in absence of quantitive assays that probe kinetics of the assembly process and and the role of parameters like pH, salt concentration and protein-nucleic acid allostery, we have seen limited progress made for anti-virals against the viral structural proteins. Our goals here are:
a) to develop techniques that allow characterization of structural dynamics of macromolecular assemblies at the nanometer scale with sub-second time resolution.
b) to probe the Dengue virus capsid assembly at the single virus level in real-time and probe the role of protein-nucleic acid allostery.

Related Articles

Zlotnick 2003: Review of Viral Assembly


Single Molecule Imaging and Spectroscopy

Tinoco 2011: A basic review of single molecule experiments and its use to dissect mechanisms is described

Joo 2006: Another review of single molecule techniques

Yildiz 2003: Demonstrates that a single molecule can be tracked with 1 nm precision

Super-Resolution Microscopy

Huang 2010: Review of Super-Resolution Microscopy

Wang 2011: Imaging of bacteria at super-resolution reveals new level of organization inside the cell


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