# BRIGHT Enables High‐SNR Live‐Cell Imaging of Non‐Repetitive Sequences via Bivalent Fluorescent Nanobody‐Mediated Cascade‐Dependent Illumination

## TL;DR

BRIGHT is a new imaging system that improves the clarity of live-cell DNA imaging, allowing detailed tracking of non-repetitive DNA sequences.

## Contribution

BRIGHT introduces a bivalent fluorescent nanobody system that significantly enhances imaging signal-to-noise ratios for non-repetitive DNA.

## Key findings

- BRIGHT achieves up to 284.84 SNR for non-repetitive DNA imaging.
- It enables tracking of chromatin dynamics and extrachromosomal circular DNA.
- BRIGHT outperforms existing tools like SunTag and PP7-PCP in SNR.

## 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.

BRIGHT is a newly developed live‐cell imaging system that integrates cascade amplification with background denoising through the bivalent binding capability and antigen‐dependent stability of a bivalent fluorescent nanobody targeting ALFA peptides. It enables high signal‐to‐noise ratio imaging of non‐repetitive DNA loci (up to 284.84), features a streamlined three‐component design, offers flexible modularity, and demonstrates broad applicability.