Live cell imaging of DNA and RNA with fluorescent signal amplification and background reduction techniques

Abstract

Illuminating DNA and RNA dynamics in live cell can elucidate their life cycle and related biochemical activities. Various protocols have been developed for labeling the regions of interest in DNA and RNA molecules with different types of fluorescent probes. For example, CRISPR-based techniques have been extensively used for imaging genomic loci. However, some DNA and RNA molecules can still be difficult to tag and observe dynamically, such as genomic loci in non-repetitive regions. In this review, we will discuss the toolbox of techniques and methodologies that have been developed for imaging DNA and RNA. We will also introduce optimized systems that provide enhanced signal intensity or low background fluorescence for those difficult-to-tag molecules. These strategies can provide new insights for researchers when designing and using techniques to visualize DNA or RNA molecules.

Spatiotemporal visualization of DNA replication by click chemistry reveals bubbling of viral DNA in virion formation

“…While FISH allows for multiplexing at high sensitivity, especially when combined with branched DNA technology (80), fluorescent reporters are applicable to both chemically fixed and live cells. Their use has remained challenging, however, due to variability in target identification, overexpression, and multimerization of up to hundreds of fluorescent tags on a target sequence (81). This can be problematic, as even small tags, such as lysinecysteine-lysine (KCK) affect target DNA compaction, for example in case of ParB and ParB binding to target DNA (82).…”

活细胞rna动态成像技术进展

“…只有互补序列与目标RNA杂交后, 荧光基团和淬 灭基团分开, 才能产生荧光 [89] (图4A). MB具有高特异性、高灵敏度、低背景的特点, 已 广泛用于成像RNA [90,91] . 2017年, 北京大学陈匡时研究 团队 [92] TBI (Broccoli) [74] 485 527 35100 0.64 71 DFHO (Corn) [57] 505 545 29000 0.25 70 DFAME (Beetroot) [62] 514 619 22500 0.17 460 OBI (Red Broccoli) [75] 541 590 47300 0.67 23 TMR-DN (RhoBAST) [65] 564 590 96000 0.57 15 SpyRho (RhoBAST) [68] 562 581 65000 0.95 34 TMR 2 (biRhoBAST) [85] 564 590 164700 0.93 0.04 的RNA分子, 实时动态示踪新生的RNA分子 [95] (图4B).…”