Under the microscope, researchers often observe different cell types that organize in peculiar patterns within tissues, or sometimes a rare cell type characterized by occupying a unique position, having an unusual shape, or expressing a particular biomarker molecule. In order to determine the deeper meaning of their observations, they have developed approaches to also access the gene expression patterns (transcriptomes) of cells by analyzing the gene-derived RNA molecules present in them, which they associate with the shapes, spatial positions and Cell molecules can match biomarkers.
However, these “spatial transcriptomics” approaches still capture only a fraction of a cell’s total RNA molecules and cannot provide the depth and quality of analysis that single-cell sequencing methods, which have been developed to study the transcriptomes of single cells, offer. isolated from tissues or biofluids above Next Generation Sequencing (NGS) techniques. They also don’t allow researchers to focus only on specific cells based on their location in a tissue, which would make the search for disjointed cell populations or rare, difficult-to-isolate cells like rare brain cells with unique functions or immune cells that attack tumors much easier . In addition, because the original tissue environment is disrupted, many spatial transcriptomics and all single-cell sequencing methods prevent researchers from revisiting their samples for follow-up analysis and are costly, requiring specialized instruments or reagents.
A new advance made at the Wyss Institute for Biologically Inspired Engineering at Harvard University now overcomes these limitations with a DNA nanotechnology-based method called “Light-Seq”. Light-Seq allows researchers to “geotag” the entire repertoire of RNA sequences with unique DNA barcodes specific to just a few cells of interest. These target cells are selected with light under a microscope above a fast and effective photocrosslinking process.
Using new DNA nanotechnology, the barcoded RNA sequences are then translated into contiguous strands of DNA, which can then be collected from the tissue sample and identified using NGS. The Light-Seq process can be repeated with different barcodes for different cell populations within the same sample, which is left intact for follow-up analysis. With performance comparable to single-cell sequencing methods, it greatly expands the depth and scope of possible studies on a tissue sample. The method is published in natural methods .
“Light-Seq’s unique combination of capabilities fulfills an unmet need: the ability to use a single instrument to perform imaging, spatially prescribed, deep sequence analysis of difficult, if not impossible, to isolate cell populations or rare cell types in preserved tissues – to one correspondence of their highly refined gene expression state with spatial, morphological, and potentially disease-related features,” said Peng Yin, Ph.D., one of four corresponding authors and a core faculty member at the Wyss Institute, where his group developed Light-Seq. “So it has the potential to advance the biological discovery process in various biomedical research areas.” Yin is also a professor of systems biology at Harvard Medical School (HMS).
From the barcode on site for sequencing ex situ
The Light-Seq project was led by Jocelyn (Josie) Kishi, Ph.D., Sinem Saka, Ph.D., and Ninning Liu, Ph.D. in Yin’s group at Wyss and Emma West, Ph.D. in Constance Cepko’s lab at HMS. Previously, Kishi and Saka had developed SABER-FISH as a spatial transcriptomics method to image gene expression directly in intact tissues (on site). “With SABER-FISH, we were still orders of magnitude away from capturing the full gene expression programs of cells with many thousands of different RNA molecules per cell. said co-first author and co-corresponding author Kishi. “Light-Seq solves this problem by combining high-resolution barcode labeling with full transcriptome sequencing via NGS, giving us the best of both worlds and additional key benefits.” At the time of the study, Kishi Wyss was a Technology Development Fellow on Yin’s team and is now pursuing a path to commercialization of Light-Seq along with some of her co-authors.
“In order to specifically sequence the cells at selected sites of intact tissue samples, we developed a new approach to photocrosslink DNA barcodes with copies of RNA molecules and a DNA nanotechnology-based method that captures them and their attached RNA sequences for NGS makes readable. said co-first author Liu, a postdoc in Yin’s group who previously co-developed a parallelized DNA barcoding platform for a high-resolution imaging method called Action-PAINT, which also became one of the core components of Light-Seq.
First, DNA primers base-pair with RNA molecules in cells and are extended to create copies of RNA sequences called complementary DNA sequences (cDNAs). Then, DNA barcode strands containing an ultrafast photocrosslinker nucleotide are in turn base-paired with the cDNAs in the cells. These are permanently linked together when a target cell is illuminated under the microscope by a stencil-like optical device that keeps other non-target cells in the microscopic field in the dark, sparing them from the photocrosslinking reaction. After washing the barcoded DNA sequences from cells that were not permanently connected on sitethe process can be repeated with different barcodes and light patterns to mark additional regions of interest.
“To be able to integrate this barcode workflow into NGS, we developed a new stitching reaction based on DNA nanotechnology. This innovation enables us to convert our barcoded cDNAs into contiguous readout sequences. We can then extract the complete collection of barcoded cDNA sequences from the sample and analyze them using standard NGS techniques,” explained Saka, one of the corresponding authors of the study, who is currently a group leader at the European Molecular Biology Laboratory in Heidelberg, Germany “Ultimately, each barcode traces the complete transcriptome display back to the preselected cells in the tissue sample, which remains intact for subsequent analyses. This offers us a unique opportunity to revisit the very same cells after sequencing for validation or further exploration.”
View of complex tissues and rare cells
After initially validating Light-Seq in cultured cells, Yin’s team wanted to apply it to a complex tissue and joined forces with the group of Constance Cepko, Ph.D. at HMS. Cepko is one of the corresponding authors of the study and Bullard Professor of Genetics and Neuroscience at HMS’s Blavatnik Institute and studies the development of the retina as a model of the nervous system. Kishi, Saka, and Liu joined West in Cepko’s group to apply Light-Seq to cross-sections of the mouse retina and profile three major layers with distinct functions. The researchers achieved sequence coverage comparable to single-cell sequencing methods and found that thousands of RNAs were enriched between the three main layers of the retina. They also showed that after sequence extraction, the tissue samples remained intact and could be further imaged for proteins and other biomolecules.
“By taking Light-Seq to extremes, we were able to isolate the full transcriptome of a very rare cell type known as ‘dopaminergic amacrine cells’ (DACs), which are extremely difficult to isolate due to their intricate connections to other cells in the retina, by retrieving only four to eight individually barcoded cells per cross-section,” West said. DACs are involved in regulating the eye’s circadian rhythm by adapting visual perception to different exposures to light during the day-night cycle. “Light -Seq also picked up RNAs specifically expressed in DACs at low levels, as well as dozens of DAC-specific biomarker RNAs that, to our knowledge, had not been previously described, opening new avenues for studying this rare cell type,” added West who was a graduate student and then postdoctoral fellow at Cepko at the time of the study and now joins Kish i in their Light-Seq commercialization efforts.
Opening up the realm of spatial transcriptomics to NGS also adds information at the level of a single RNA species. “Our sequencing data clearly demonstrated that Light-Seq can determine natural variations in the structure of RNAs. In the future, we are very interested in using Light-Seq to better understand the interplay between the immune system, disease-spreading cells, and other therapeutic strategies such as gene and cell therapy,” said Kishi.
The Light-Seq technology developed in Peng Yin’s group in the Wyss Institute’s Molecular Robotics Initiative shows once again how taking a completely unconventional approach and leveraging synthetic biology can result in a disruptive technology with great potential to transform both basic research and research as well as advancing clinical medicine.”
Donald Ingber, MD, Ph.D., Founding Director of Wyss
Donald Ingber is that too Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital and the Hansjörg Wyss Professor of Bio-Inspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
Wyss Institute for Biologically Inspired Engineering at Harvard
kish, JY, et al. (2022) Light-Seq: In situ light-driven barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing. natural methods. doi.org/10.1038/s41592-022-01604-1.