Cornell University

Yale University

James Munro

Scott C Blanchard

Daniel Terry

Walther Mothes

Peter Geggier

A modified HIV gp120 protein labeled with a fluorophore has been used herein in single-molecule imaging techniques to demonstrate the conformation states of HIV-1 Env. The introduction of small organic fluorophores at selected positions within HIV-1 envelope protein gp120 that do not affect infectivity permits the detection of changes in inter-dye distances as a measure of conformational changes. Implementation of the smFRET-based technologies disclosed herein enable conformational screening for molecules that block, induce or trap HIV-1 Env in specific conformational states. Methods for identifying anti-HIV drugs in a smFRET-based screening, and various reagents useful for implementation of such methods, are provided.

FRET-based reagents and methods for identifying anti-HIV compounds

Automatically selecting time traces from a fluorescence experiment, in one aspect, may include capturing results of the fluorescence experiment in a moving image; localizing sources of fluorescence in the moving image; producing time traces of each fluorescent source by monitoring fluorescence intensity of said localized sources in the moving image over time; removing unuseful time traces from said produced time traces; and selecting useful time traces from said produced time traces based on one or more defined criteria. FRET traces from selected time traces may be further calculated and analyzed. A unified computer-implemented platform in one aspect may include tools to locate single molecules, extract traces, classify smFRET traces according to adjustable parameters, and quantify the kinetic parameters of FRET transitions using analytical procedures such as Hidden Markov Modeling (HMM) procedures.

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New Haven, CT, United States of America

James Munro