Rahul Roy

A Practical Guide to Single Molecule FRET

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

Single-molecule imaging of transcription factor binding to DNA in live mammalian cells

Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.

Z 2 classification of quantum spin Hall systems: An approach using time-reversal invariance

We study the phases of Bloch insulators with time-reversal symmetry on the basis of the homotopy of the ground-state wave functions in momentum space and find that there are two topological classes characterized by a

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

{Z}_{2}

Edge modes, edge currents, and gauge invariance inpx+ipysuperfluids and superconductors

invariant. The results are in agreement with a recent study based on counting the zeroes of a certain Pfaffian function related to the ground-state wave function. It is shown that there is a link between the formulation of the topological invariant presented here and the number of robust edge states. A formula is also provided which greatly simplifies the computation of the invariant in a large number of cases. The present study provides guidance for the search of systems which belong to the nontrivial topological class and also establishes a link between the quantum spin Hall effect and the integer quantum Hall effect.

Transcription Initiation in a Single-Subunit RNA Polymerase Proceeds through DNA Scrunching and Rotation of the N-Terminal Subdomains

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA–RNA interactions.

Abelian Floquet symmetry-protected topological phases in one dimension

The excitation spectrum of a two-dimensional

Periodic table for Floquet topological insulators

{p}_{x}{+ip}_{y}

Collective modes and electromagnetic response of a chiral superconductor

fermionic superfluid, such as a thin film of

Band geometry of fractional topological insulators

{}^{3}\mathrm{He}\ensuremath{-}A,