Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Single-molecule imaging techniques have been applied herein and have demonstrated the conformation states of HIV-1 Env. The introduction of small organic fluorophores at selected positions within HIV-1 Env 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Surface-bound, translationally competent ribosome complexes are used to generate a translation profile for mRNA, which mRNA may be a single molecular species, or a combination of species, including complex mixtures such as those found in the set of mRNAs isolated from a cell or tissue. One or more components of the surface-bound ribosome complex may be labeled at specific positions to permit analysis of multiple or single molecules for determination of ribosomal conformational changes and translation kinetics. Translation profiles are used as the basis for comparison of an mRNA or set of mRNA species. The translation profile can be used to determine such characteristics as kinetics of initiation, kinetic of elongation, identity of the polypeptide product, and the like. Analysis of translation profiles may be used to determine differential gene expression, optimization of mRNA sequences for expression, screening drug candidates for an effect on translation, etc.

Abstract: 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; and the like. 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.