Publication

A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

replaced
   April 26th, 2018 at 8:23pm

Note: Replaced Biorxiv  


This biorxiv set was replaced by PMID:30858596.

Overview


Abstract

Cellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

Authors

Rajarshi P Ghosh  •  J Matthew Franklin  •  Will E. Draper  •  Quanming Shi  •  Jan T. Liphardt

Link

https://www.biorxiv.org/content/10.1101/159004v1


Journal

bioRxiv

doi:10.1101/159004

Published

July 3rd, 2017