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