NEW YORK – Researchers from the Massachusetts Institute of Technology and their collaborators have developed a new assay that can directly measure nascent RNAs produced during gene expressions at the single-cell level.

纽约——麻省理工学院的研究人员及其合作者开发了一种新的检测方法，可以直接测量单细胞水平上基因表达过程中产生的新生RNA。

Described in an article in Nature last month, the method, coined single-cell global run-on and sequencing (scGRO-seq), offers researchers a fuller picture of transcription events and enhancer-gene dynamics for individual cell types that are difficult to achieve with other methods.

上个月在《自然》杂志的一篇文章中描述了这种方法，这种方法被称为单细胞全局运行和测序（scGRO-seq），它为研究人员提供了一个更全面的转录事件和增强子基因动力学的图片，这些都是其他方法难以实现的。

'A precise way of looking at active enhancers in a given cell type has been of interest for many people for a long time,' said Dig Mahat, a postdoctoral researcher in Phillip Sharp’s lab at MIT and the first author of the study. 'This tool could unlock various ways to studying gene regulation, and potentially even have a role in understanding diseases.' .

麻省理工学院菲利普·夏普实验室的博士后研究员、该研究的第一作者迪格·马哈特（DigMahat）说，长期以来，许多人一直对在特定细胞类型中观察活性增强子的精确方法感兴趣这个工具可以打开研究基因调控的各种方法，甚至可能在理解疾病方面发挥作用。”。

Nascent RNAs — RNA molecules that are in the process of being synthesized and attached to RNA polymerase — allow researchers to more comprehensively investigate gene expression events and study unstable transcripts, such as those from active enhancers, that are otherwise difficult to detect.

新生RNA-正在合成并附着在RNA聚合酶上的RNA分子-使研究人员能够更全面地研究基因表达事件，并研究不稳定的转录本，例如活性增强子的转录本，否则很难检测到。

'The beauty of studying nascent RNAs is that we are capturing the molecules as they are being made,' Mahat said. 'By looking at [those molecules], we get a full picture of what gene is being transcribed at that moment.'

马哈特说：“研究新生RNA的美妙之处在于，我们正在捕获正在制造的分子。”通过观察（这些分子），我们可以全面了解当时转录的基因。”

Previous methods for bulk nascent RNA sequencing, such as global run-on and sequencing (GRO-seq) and precision run-on and sequencing (PRO-seq), enabled simultaneous quantification of transcription in genes and enhancers. However, these assays cannot measure nascent RNAs from individual cells, leaving out important biological insights into enhancers, which are highly specific to cell types and states, the study authors noted.

先前用于批量新生RNA测序的方法，例如全局运行和测序（GRO-seq）和精确运行和测序（PRO-seq），可以同时定量基因和增强子中的转录。然而，研究作者指出，这些检测方法无法测量单个细胞的新生RNA，从而遗漏了对增强子的重要生物学见解，增强子对细胞类型和状态具有高度特异性。

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Meanwhile, conventional single-cell methods are designed to capture mRNAs by their poly(A) tail, making them unsuitable for measuring nascent RNAs from genes and enhancers, which are not polyadenylated and tend to be unstable. Because most nascent RNAs degrade rapidly after they are made, the traditional scRNA-seq methods cannot offer 'the most up-to-date picture' of transcription that is happening in a cell, Mahat noted..

同时，传统的单细胞方法被设计为通过其poly（A）尾巴捕获mRNA，使其不适合测量来自基因和增强子的新生RNA，这些基因和增强子不是聚腺苷酸化的，并且往往不稳定。Mahat指出，由于大多数新生RNA在制造后会迅速降解，因此传统的scRNA-seq方法无法提供细胞中发生的转录的“最新图片”。。

One of the biggest challenges in developing a nascent RNA sequencing method for single cells was to capture sufficient nascent RNA molecules, which can be scarce in an individual cell.

开发单细胞新生RNA测序方法的最大挑战之一是捕获足够的新生RNA分子，而这些分子在单个细胞中可能很少。

'Handling this small amount of RNA from single cells was challenging,' Mahat noted. 'For the longest time, we assumed that it would be almost impossible to do single cell for nascent RNA sequencing.'

“处理来自单个细胞的少量RNA具有挑战性，”马哈特指出在很长一段时间内，我们认为几乎不可能用单细胞进行新生RNA测序。”

To overcome this technical barrier, the MIT researchers devised a new strategy that can selectively label nascent RNAs through a nuclear run-on reaction using modified nucleotide triphosphates (NTPs) and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), known as click chemistry.

为了克服这一技术障碍，麻省理工学院的研究人员设计了一种新策略，可以通过使用修饰的核苷酸三磷酸（NTP）和铜（I）催化的叠氮化物-炔烃环加成（CuAAC）的核连续反应选择性标记新生RNA，称为点击化学。

Intact nuclei containing nascent RNA are first labeled with propargyl using 3′-(O-propargyl)-NTPs. After that, they are sorted into individual wells in a 96-well plate, each containing denaturing reagents and unique DNA barcodes. Subsequently, the barcoded nascent RNAs are pooled, reverse transcribed, PCR amplified, and sequenced.

。之后，将它们分类到96孔板中的各个孔中，每个孔都包含变性试剂和独特的DNA条形码。随后，合并条形码化的新生RNA，逆转录，PCR扩增和测序。

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Mahat said the team has spent a lot of effort on optimizing the workflow, including identifying chain-terminating nucleotides that are compatible with the polymerase and the click chemistry, as well as finding the best reaction conditions for reverse transcription and PCR amplification.

Mahat说，该团队在优化工作流程方面付出了大量努力，包括鉴定与聚合酶和点击化学相容的链终止核苷酸，以及寻找逆转录和PCR扩增的最佳反应条件。

For the published study, the researchers applied scGRO-seq to analyze genes and enhancers across 2,635 individual mouse embryonic stem cells. Overall, they demonstrated the method's ability to directly profile the mechanisms of transcription regulation and the role of enhancers in gene expression, enabling the analysis of transcription kinetics at the single-cell level..

对于已发表的研究，研究人员应用scGRO-seq分析了2635个小鼠胚胎干细胞的基因和增强子。总体而言，他们证明了该方法能够直接分析转录调控机制和增强子在基因表达中的作用，从而能够在单细胞水平上分析转录动力学。。

'This is a very interesting paper,' said Junyue Cao, head of the single-cell genomics and population dynamics laboratory at Rockefeller University, who was not involved in the study.

“这是一篇非常有趣的论文，”洛克菲勒大学单细胞基因组学和种群动力学实验室负责人曹俊岳说，他没有参与这项研究。

Calling scGRO-seq 'a great improvement' to its bulk predecessor, Cao said he believes the method, which is the first of its kind in the field, will enable many applications, such as characterizing transcriptional burst, analyzing transcriptional activities of cell cycle-related nonconventional genes, and studying interactions between enhancers and genes..

Cao称scGRO-seq是对其大量前身的“巨大改进”，他认为该方法是该领域的第一种方法，将实现许多应用，例如表征转录爆发，分析细胞周期相关非常规基因的转录活性，以及研究增强子和基因之间的相互作用。。

Despite its promises, Cao also noted some current limitations of the approach. For one, he said the capture efficiency is still not very high, leaving room for future improvements. In addition, since the study only tested the method in mouse embryonic stem cells, it remains to be seen how it will perform in other organisms or tissues, especially during in vivo transcriptome profiling.

尽管做出了承诺，曹还指出了该方法目前的一些局限性。首先，他说捕获效率仍然不高，为未来的改进留下了空间。此外，由于该研究仅在小鼠胚胎干细胞中测试了该方法，因此它在其他生物体或组织中的表现仍有待观察，尤其是在体内转录组分析过程中。

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Echoing Cao’s point, Mahat said that 'there is a lot of room for improvement' when it comes to the capture efficiency of scGRO-seq, which can only detect about 10 percent of nascent RNAs within a cell at the moment.

Mahat回应了Cao的观点，他说，就scGRO-seq的捕获效率而言，“还有很大的改进空间”，目前scGRO-seq只能检测到细胞内约10%的新生RNA。

Moreover, Mahat acknowledged that the throughput of the method is still pretty low given it is carried out on a 96-well plate. Throughput could be improved by switching to a droplet-based method, he noted.

此外，Mahat承认，鉴于该方法是在96孔板上进行的，因此该方法的通量仍然很低。他指出，可以通过切换到基于液滴的方法来提高吞吐量。

Down the road, he and his collaborators are also hoping to measure not only nascent RNAs but also mature transcripts at the same time.

在这条路上，他和他的合作者也希望不仅测量新生的RNA，而且同时测量成熟的转录本。

A US patent pertaining to scGRO-seq has already been granted to MIT, with Mahat and Sharp, who is the paper’s corresponding author, as inventors. Mahat said the team is in contact with a few single-cell companies for licensing the IP for kit development.

与scGRO-seq相关的美国专利已经授予麻省理工学院，论文的通讯作者马哈特和夏普作为发明人。Mahat说，该团队正在与几家单细胞公司联系，以获得用于试剂盒开发的IP许可。

Mahat said one of his goals is to use scGRO-seq to establish a cell-specific enhancer-gene map, elucidating the role of enhancers in disease development.

马哈特说，他的目标之一是使用scGRO-seq建立细胞特异性增强子基因图谱，阐明增强子在疾病发展中的作用。

'Having the map in various cell types, various conditions, and various tissue types, we will be much closer to understanding what these enhancers and their associated variants are contributing to diseases,' he said. 'I think scGRO-seq is going to be a major tool in revealing that map, and we're excited about that.' .

他说：“拥有不同细胞类型、不同条件和不同组织类型的图谱，我们将更接近了解这些增强子及其相关变体对疾病的贡献。”我认为scGRO-seq将成为揭示地图的主要工具，我们对此感到兴奋。”。