Cornell University

Leland Stanford Junior University

University of California

Columbia University

Yale University

University of Innsbruck

Leopold-franzens-universität Innsbruck

Scott C Blanchard

Roger Altman

J David Warren

Ronald Micura

Andrea Haller

James Munro

Michael Brian Feldman

Leyi Wang

Brian K Kobilka

James Harrison Doudna Cate

Jonathan A Javitch

Joseph Puglisi

Daniel Terry

Harel Weinstein

Arto Pulk

Michael R Wasserman

Walther Mothes

Ruben L Gonzalez

Gabriel Glenn Gregorio

Zhou Zhou

Peter Geggier

Marie F Souliere

Disclosed herein are methods for providing a molecular efficacy of a ligand, especially when utilizing single-molecule fluorescence resonance energy transfer ('smFRET') imaging, as well as compounds useful in such methods.

Methods for quantifying ligand efficacy in G-protein coupled receptors using single-molecule fluorescence energy transfer

This disclosure is directed to methods of conducting dynamic single-molecule fluorescence studies such as smFRET on a membrane protein which permits observation and quantification of conformational dynamics of a membrane protein. Also disclosed herein are mutant membrane proteins in which one or more mutations have been introduced for affixing a fluorophore, as well as reagents and kits containing such mutant membrane proteins for conducting dynamic single-molecule fluorescence studies. The methods and compositions disclosed herein can be used in screening for compounds that enhance or reduce the activity of a membrane protein, useful for treating diseases associated with the malfunction of the membrane protein or alterations in membrane protein conformation.

SmFRET with membrane proteins

We provide isolated TPP and preQ1 class II riboswitches which are labelled for FRET studies of ribosome function. The riboswitches may be used in assays to determine riboswitch function, and to test the activity of compounds in modulating riboswitch function.

Methods and reagents for riboswitch analysis

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

The invention is directed to fluorophore-containing compositions and configurations wherein proximity between the fluorophore and one or more protective agents (PAs) modifies the lifetime of fluorescent and/or dark states, their frequency of occurrence, and the total lifetime of fluorescence in order to appropriately modify the photophysical characteristics of the fluorophore. The invention is also directed to methods that utilize these compositions and configurations.

Methods and compositions for altering photophysical properties of fluorophores via proximal quenching

Dye compounds of the formula (1) wherein A is a protective agent group that has a characteristic of modifying the singlet-triplet occupancy of the shown cyanine moiety, and M is a reactive crosslinking group or a group that can be converted to a reactive crosslinking group. Methods for synthesizing the dye compounds and applications for their use are also described. 

Dye compositions, methods of preparation, conjugates thereof, and methods of use

The present invention describes the synthesis of biological samples that can be used for the purpose of enhancing the signal-to-noise ratios achievable during the imaging of protein synthesis, amino acid transport and neurotransmitter transport, particularly in applications where single-molecule resolution is demanded. The present invention provides quencher-labeled elongation factor (EF-Tu) and fluorophore-labeled tRNA. When these molecules are present in a ternary complex with GTP, the fluorescently-labeled tRNA is quantitatively quenched. Once the tRNA is incorporated into an actively translating ribosome, however, a burst of fluorescence is released and can be detected by a variety of techniques, including smFRET imaging. The invention further provides novel EF-Tu constructs for achieving quencher labeling at high levels while retaining native or near native activity in the translation reactions, as well as methods for preparing stable ternary complexes, methods of protein sequencing, methods of detecting amino acid transport using a proteoliposome assay system and the proteoliposomes systems and methods of imaging translation events in single living cells. The present invention should have an immediate impact on next-generation sequencing technologies and the detection of neurotransmitter transporter activities in both in vitro and in vivo settings, a critical component of drug activity/screening assays targeting this important class of molecules.

Signal amplification methods for the imaging of protein synthesis and neurotransmitter transport

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.

Computer-implemented platform for automated fluorescence imaging and kinetic analysis

The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.

Ribosomal targets for antibiotic drug discovery

The present invention is provides isolated riboswitches with FRET pairs for distinguishing changes in regulatory interactions controlled by the expression platform domain found in riboswitches. The invention further provides methods of using those riboswitches to detect structural changes in the expression platform domain and to identify potential antibiotics.

Methods and reagents for analyzing riboswitches using FRET

This invention relates to a method to rapidly find crystallization conditions for a biomolecule in a desired conformation using single-molecule, fluorescent resonance energy transfer (smFRET) imaging techniques. The method provides significant cost and time advantages over the empricial exploration for crystallization conditions.

Single molecule imaging techniques to aid crystallization

Translationally competent ribosomes are bound to a solid substrate through a specific attachment site. A spatial array of such immobilized ribosomes may be produced on a planar substrate, microbeads, on fiber optics. One or more components of the ribosome complex are preferably labeled, e.g. with a fluorescent dye. Ribosomal RNAs, including mRNA and tRNA; and/or ribosomal proteins may be labeled at specific positions, and arrays of immobilized ribosomes may comprise a panel of different labels and positions of labels. Fluorescence detection can then be used to monitor conformational dynamics and translation rates.

Growing community of inventors

Ithaca, NY, United States of America

Scott C Blanchard