Cornell University

University of California

University of Innsbruck

Roger Altman

Scott C Blanchard

J David Warren

Leyi Wang

Michael Brian Feldman

James Harrison Doudna Cate

Ronald Micura

Andrea Haller

Marie F Souliere

Arto Pulk

Michael R Wasserman

Zhou Zhou

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

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

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

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.

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Roger Altman