[R35] Structure, Accessibility and Extension of Telomeric Overhangs-Supplement for a Confocal Microscope

Ente: National Institute of General Medical Sciences

Scadenza: 2030-01-31

Importo max: $245,000

Paese: US

Descrizione

Project Summary: We request Administrative Supplement funds to purchase an EI-FLEX Pro microscope from Exciting Instruments. Our R35 grant focuses on: (1) Length dependent variation of structural and kinetic characteristics of telomeric overhangs (ssTEL); (2) The impact of Shelterin, telomeric repeat containing RNA (TERRA), and small molecules on accessibility of ssTEL to nucleic acid probes, DNA damage response activators, nucleases, and telomerase; (3) How relevant physiological factors affect telomerase-catalyzed telomere extension and the folding patterns of newly synthesized ssTEL. We study these questions using primarily single molecule methods that rely on surface immobilization of molecules. While these methods have been instrumental in monitoring ssTEL and their interacting partners for extended periods, they require laborious surface treatment to minimize non-specific interactions with the surface. Also, surfaces need to be modified to enable immobilization of molecules within the penetration depth of total internal reflection beam. Finally, it is necessary to create microfluidic chambers for buffer exchange and to ensure effective function of oxygen scavenging system, critical for photostability, over extended imaging periods. For a typical experiment, 30-50 l solution at 1-100 nM concentration is required. The imaging is performed by an EM-CCD and time resolution is typically 30 ms or longer. Here, we request funds to purchase an EI Flex Pro microscope, which can perform single molecule FRET, alternating laser excitation (ALEX) and Fluorescence (Cross) Correlation Spectroscopy (FCS/FCCS) measurements in a confocal mode. Single molecule detection is accomplished using a single drop of liquid (a few l) placed on a coverslip. Molecules that are ~10 m away from the surface are detected while they diffuse through the laser beam for fraction of a second. While the capability of attaining kinetic information over extended periods is compromised, this mode of detection: (i) eliminates the need for the laborious surface treatment and immobilization; (ii) requires an order of magnitude less volume and an order of magnitude less biomolecule concentration; (iii) eliminates the need for oxygen scavenging system since molecules are excited for less than a second; (iv) enables signal detection with ~1 ms time resolution. The fluorescence bursts that are detected as the fluorescently labeled biomolecules diffuse through the focused laser beam can be analyzed to attain molecular distances via smFRET, binding stoichiometry via FCS and ALEX, and diffusion coefficients and hydrodynamic radii via FCS. The instrument has built-in analysis modules for all these capabilities and provides a practical and user friendly interface where analysis can be performed in real-time, during data acquisition. This instrument perfectly complements our existing capabilities and will enable us to utilize the know-how and experience we have accumulated about telomeres and Istituzione: KENT STATE UNIVERSITY PI: Hamza Balci Progetto: 3R35GM156183-02S1

Settori: National Institute of General Medical Sciences

Vai al bando originale

Registrati gratis su Bandolo per trovare bandi compatibili con la tua azienda.