Howard Hughes Medical Institute

The Brigham and Women's Hospital, Inc.

New York University

Eric R Schreiter

Loren Lee Looger

Ahmed Abdelfattah

Richard Theodore Lee

Luke D Lavis

Jonathan S Marvin

Douglas S Kim

Karel Svoboda

Benjamin F Fosque

Claire Deo

Hersh Bhargava

Jonathan B Grimm

Dmitry Rinberg

Ilya Kolb

Fern Sha

Laurel M Heckman

Yan Zhang

Jeremy P Hasseman

A chemigenetic calcium indicator and a method of measuring calcium are provided. The chemigenetic calcium indicator includes a calcium-binding protein domain attached to a ligand binding protein domain. The method of measuring calcium includes administering a chemigenetic calcium indicator to a subject and determining changes in fluorescence, the chemigenetic calcium indicator including a ligand binding protein domain having a calcium-binding protein domain and a dye-ligand conjugate attached thereto.

Chemigenetic calcium indicators

A voltage indicator includes a polypeptide sequence comprising a voltage-sensitive opsin domain and a capture protein domain arranged and disposed to capture a fluorescent dye ligand. When the fluorescent dye ligand is captured and the voltage indicator is bound to a cell membrane, an increase in voltage across the cell membrane causes an increase in fluorescent emission.

Voltage indicators

Reversibly switchable fluorescent protein-based indicators are disclosed, and can be used as neuronal activity markers. The disclosed reversibly switchable fluorescent protein-based indicators exhibit faster or slower photoswitching the presence or absence of calcium, and depending on the wavelength of light stimulus employed.

Reversibly switchable fluorescent protein-based indicators

Genetically encoded calcium indicator (GECI) polypeptides and the nucleic acid molecules encoding such polypeptides are provided.

Genetically encoded calcium indicators (GECIs) and methods of making and using

Genetically encoded calcium indicator (GECI) polypeptides and the nucleic acid molecules encoding such polypeptides are provided. In addition, methods of using such nucleic acids and polypeptides in methods of screening for agonists or antagonists of G-protein coupled receptor (GPCR) or ion channels and methods of monitoring neural activity also are provided.

Genetically encoded calcium indicators and methods of use

Provided herein are a voltage indicator and a method of measuring voltage. The voltage indicator includes a membrane-localized voltage-sensitive protein coupled to a capture protein. The method of measuring voltage includes administering a voltage indicator including a membrane-localized voltage-sensitive protein coupled to a capture protein, and determining changes in fluorescence of a small-molecule fluorescent dye captured by the capture protein.

Chemigenetic voltage indicators

The presently-disclosed subject matter relates to analyte-activated photolabile compounds. The compounds include the formula: wherein Z includes a maskable molecule; L is selected from a bond, C, C(O), O, alkyl, (O)alkyl, and alkoxy; Ris selected from H, halogen, alkyl, and acyl; each Ris independently selected from H, alkyl, aryl, and acyl, and is optionally substituted with one or more heteroatoms independently selected from COOH and COO(alkyl); and Ris selected from H, alkyl, aryl, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, CHO, COOH, COO(alkyl), COO(aryl), POH, and SOH, and is optionally substituted with one or more heteroatoms independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO, CHO, COO, COOH, COO(alkyl), COO(aryl), C(O)NR, POH, and/or SOH. Also provided are methods of detecting calcium and treating a subject with the analyte-activated photolabile compounds.

Ion-activated photolabile compounds

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

Genetically encoded biosensors

The presently-disclosed subject matter includes fluorescent protein-based indicators for detecting ions, small molecule analytes, or combinations thereof. In some embodiments the indicators include a polypeptide, which itself includes a fluorescent polypeptide, a compound-binding polypeptide, and a polypeptide target of the compound-binding polypeptide. In some embodiments the polypeptide includes an EosFP polypeptide, a calmodulin polypeptide, and a M13 polypeptide, or fragments and/or variants thereof. The presently-disclosed subject matter also includes methods for detecting calcium in a sample with embodiments of the present polypeptides. In some embodiments the present indicators experience a permanent shift from green to red fluorescent when exposed to an detecting substance, such as calcium.

Methods of using fluorescent protein-based indicators

Protein indicators useful for calcium imaging, in particular, red genetically-encoded calcium indicators (GECIs) disclosed herein rival best-of-class green GECIs in terms of sensitivity for detecting neural activity, and can be monitored in vivo. The presently-disclosed subject matter further includes a method of monitoring cell activity comprising stimulating a cell comprising a red GECI polypeptide; and detecting fluorescence emitted by the cell.

Red genetically encoded calcium indicators and methods of use

This disclosure provides methods for identification of olfactory sensosry neurons (OSN) that recognize specific odorants. The method comprises introducing into the OSN, a permanent activity marker which exhibits a detectable and permanent change upon activation of the neuron in response to an odorant and exposure to an exteranal stimulus, such as light. The OSN can be isolated and its receptor characterized.

Growing community of inventors

Ashburn, VA, United States of America

Eric R Schreiter