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Lithuanian Team Developing Droplet-Based Single-Cell Small RNA Sequencing Approach

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NEW YORK – A European research project aims to develop a high-throughput, droplet-based method for simultaneous microRNA and mRNA capture and sequencing in single cells.

The researchers, based at Vilnius University in Lithuania, want to apply the technique to assess the roles of miRNAs in cell fate decision during hematopoietic development at a single-cell level. The European Union has backed the project, called Droplet-Small-Seq, with a €146,000 ($174,000) Horizon 2020 research grant. It is set to commence Oct. 1 and run through September 2023.

According to Linas Mažutis, principal investigator of the microtechnologies group at VU's Institute of Biotechnology, while droplet-based, single-cell RNA sequencing technologies are widely used in the life sciences, most rely on the capture of polyadenylated RNA and are thus restricted to protein-coding mRNAs, meaning that other parts of the transcriptome, including small, noncoding RNAs, remain unexplored terrain for scientists.

Due to this, little is actually known about how noncoding RNAs are expressed and function at the cellular level, particularly the roles they play in cellular phenotypic diversity. Of the types of small, noncoding RNAs, miRNAs are of great interest, he noted, and are involved in the development of nearly all tissues, yet are most often interrogated using cell-based assays.

The investigators' solution therefore is to develop a droplet-based technique to sequence both small, noncoding RNAs and mRNAs at the same time.

"Many fundamental questions can and have been answered without looking into small noncoding RNAs," acknowledged Mažutis. "However, [small, noncoding RNAs] do play an important regulatory role and therefore understanding how RNA regulates itself, or how small, noncoding RNAs regulate mRNA is an interesting problem to solve," he said.

Mažutis' group is well-positioned to address such a challenge and developing technologies to support high-throughput, multiomic studies of single cells is a continuous goal. The Lithuanian investigators have already developed methods for single-cell barcoding and sequencing using droplet microfluidics, an approach profiled in a 2017 Nature Protocols paper. They have also worked on high-throughput screening of antibody-secreting cells, in vitro directed evolution using artificial cells, single-cell genome amplification, and other endeavors.

"Over the years, we have built extensive know-how and learned many cooking tips on how to make use of droplet microfluidic technology," commented Mažutis. The lab also has ongoing collaborations with partners at Harvard University, Memorial Sloan Kettering Cancer Center, and ETH Zürich.

"Most of our collaborations involve microfluidics but for different applications: from single-cell transcriptomics to directed evolution," said Mažutis. "We have extensive expertise in molecular barcoding, clinical sample dissociation, and processing, and I think the researchers we collaborate with find our expertise useful."

Mažutis noted that there will be several challenges to implementing the current research project. Small, noncoding RNAs only constitute between 1 and 3 percent of total RNA, and their diminutive size and diverse sequences make them difficult to both capture and sequence.

"Simultaneously capturing small RNAs and mRNA from the same cell technically should require multi-step reactions, making the entire process labor intensive and costly," said Mažutis. Yet to be compatible with high-throughput, droplet-based assays, small and long RNA capture has to rely on a one-step reaction, the development of which is funded under the current EU grant.

Mažutis declined to discuss the planned method at length but said it would rely on using droplet technology to capture small, noncoding RNAs. "We will synthesize DNA-barcoding hydrogel beads that will simultaneously capture both long mRNAs and short, noncoding RNAs," he said. "Once captured, the RNAs will be tagged with cell barcodes and the [unique molecular identifiers] will be amplified and sequenced."

The successful development of a high-throughput method for the simultaneous capture and sequencing of small RNAs and long mRNA from the same cells would "already be a big step forward in the field," Mažutis said, adding that such a technique could be applied in other biological systems, such as in microorganisms whose RNA molecules lack polyadenylation.

It could also advance understanding of the regulatory role of miRNAs, especially how the molecules are driving cell differentiation. "We also do not know which miRNAs are specific to a given cell type, and if so, what are their targets," he said.

Mažutis' colleague, Simonas Juzėnas, will be the lead researcher of the project. Juzėnas, who is currently on paternity leave, could not be reached for comment. But Mažutis said that his team is interested in using the developed method to explore a variety of questions. For instance, they would like to understand if miRNAs play a role in regulating the robustness of the cellular state by calibrating expression levels of driving genes, or if they drive cell fate choices by suppressing transcription factors, leading to lineage commitment.

Mažutis stressed that the project is in its infancy and declined to speculate about broader application, perhaps via a partner. "Only the results will show if it will have commercial value," he said. "It's too early to say anything at this stage."

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