Liu Yifang, Qiu Yuchen, Nian Keqing, Rouhanifard Sara H
Department of Bioengineering, Northeastern University, Boston, MA, USA.
BMC Genomics. 2025 Jul 1;26(1):624. doi: 10.1186/s12864-025-11818-0.
Transcriptional bursts are periods when RNA polymerase interacts with a DNA locus, leading to active gene transcription. This bursting activity can vary across individual cells, and analyzing the differences in transcription sites can help identify key drivers of gene expression for a specific target RNA. Scaffolding methods based on fluorescence in situ hybridization (FISH) have been widely used to amplify the fluorescent signal of RNAs and sort cells based on RNA expression levels. Examples include click-amplifying FISH (clampFISH) and hybridization chain reaction (HCR). However, these methods are limited in their ability to target and amplify transcription sites, due to the long probes' hindered accessibility through cellular compartment membranes and crosslinked proteins. Thus, sorting based on transcriptional bursting has not been achieved. Additionally, the required formaldehyde fixation interferes with downstream analysis of chromatin and protein-binding interactions.
To address these challenges, we developed a platform, nuclear clampFISH (nuclampFISH) that integrates click-amplified FISH with reversible crosslinkers and allows access to the nucleus. We demonstrate that with optimized parameters and by eliminating the cytosol and cell membrane, this method enables the amplification of fluorescent signal for RNAs using a reversible crosslinker, enabling the sorting of cells based on nuclear RNA expression and is compatible with downstream biochemical analysis including chromatin conformation assays. We applied this assay to demonstrate that transcriptionally active cells have more accessible chromatin for a respective gene.
This new method enables the sorting of cells based on transcriptional bursts. This method combines the specificity of a single-cell assay for detecting transcription sites with the throughput of flow cytometry to enable bulk assays such as chromatin conformation or other biochemical assays. Notably, the tools developed are highly accessible and do not require specialized computation or equipment.
转录爆发是RNA聚合酶与DNA位点相互作用导致基因活跃转录的时期。这种爆发活动在单个细胞间可能有所不同，分析转录位点的差异有助于识别特定靶RNA基因表达的关键驱动因素。基于荧光原位杂交（FISH）的支架方法已被广泛用于放大RNA的荧光信号，并根据RNA表达水平对细胞进行分选。例子包括点击扩增FISH（clampFISH）和杂交链式反应（HCR）。然而，由于长探针难以穿过细胞膜和交联蛋白进入细胞区室，这些方法在靶向和放大转录位点方面能力有限。因此，尚未实现基于转录爆发的分选。此外，所需的甲醛固定会干扰染色质和蛋白质结合相互作用的下游分析。
为应对这些挑战，我们开发了一个平台——核clampFISH（nuclampFISH），它将点击扩增FISH与可逆交联剂相结合，并能进入细胞核。我们证明，通过优化参数并去除细胞质和细胞膜，该方法能够使用可逆交联剂放大RNA的荧光信号，实现基于核RNA表达的细胞分选，并且与包括染色质构象分析在内的下游生化分析兼容。我们应用该检测方法证明转录活跃的细胞对于相应基因具有更易接近的染色质。
这种新方法能够基于转录爆发对细胞进行分选。该方法将用于检测转录位点的单细胞检测的特异性与流式细胞术的通量相结合，以实现诸如染色质构象或其他生化检测等大量检测。值得注意的是，所开发的工具易于使用，不需要专门的计算或设备。
