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Published on: August 6, 2014
BRIGHT Enables High-SNR Live-Cell Imaging of Non-Repetitive Sequences via Bivalent Fluorescent Nanobody-Mediated
Lei Feng1,2,3, Tao Huang1,2,3, Yanxi Han1,2,3
1National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/ National Center of Gerontology, Beijing, 100730, P. R. China.
Abstract:
Genomic loci and genome-independent DNA exhibit heterogeneous dynamics related to physiological function. Live-cell imaging of non-repetitive sequences is essential but limited by low signal-to-noise ratio (SNR), restricting accurate identification and dynamic tracking. Here, the development of a BRIGHT (bivalent near-infrared nanobody-mediated cascade illumination of genomic loci for high-SNR tracking) system for non-repetitive sequence live imaging is reported. BRIGHT employs dCas9-n×ALFA to target genomic loci and realizes high SNR by triggering cascade-dependent illumination via bivalent binding and antigen-dependent illumination of a bivalent near-infrared fluorescent nanobody targeting ALFA tags (Bi-NIR-FbALFA). BRIGHT achieves ≈6.26-fold and ≈6.76-fold higher SNR than SunTag and PP7-PCP in telomere labeling, and enables non-repetitive DNA imaging with SNR up to 284.84. Furthermore, BRIGHT tracks chromatin dynamics comparably to previous tools, links dynamics and nuclear positioning to transcriptional activity, and detects the distribution and dynamic heterogeneity of extrachromosomal circular DNA (eccDNA). Overall, BRIGHT dramatically improves the SNR of non-repetitive sequence imaging and offers an innovative tool for studying genomic dynamics and gene regulation.
