Focus on tissue cells so RNA can tell their story

Under a microscope, researchers often observe different types of cells organizing themselves in particular patterns in tissues, or sometimes a rare type of cell that stands out by occupying a unique position, having an unusual shape, or expressing a specific biomarker molecule. . To determine the deeper meaning of their observations, they developed approaches to also access the gene expression patterns of cells (transcriptomes) by analyzing gene-derived RNA molecules present within them, which they can match with the shapes, spatial and molecular positions of the cells. 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 provided by single-cell sequencing methods, which have been developed to study the transcriptomes of single cells isolated from tissues. or biofluids Going through 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 it much easier to track disjunct cell populations, or rare and difficult-to-isolate cells like rare brain cells with unique functions, or immune cells. cells that invade tumors. Additionally, 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 they are expensive because they require instruments or specialized reagents.

A new advance made at Harvard University’s Wyss Institute for Biologically Inspired Engineering now overcomes these limitations with a DNA nanotechnology-based method called “Light-Seq”. Light-Seq allows researchers to “geotag” the full repertoire of RNA sequences with unique DNA barcodes exclusive to a few cells of interest. These target cells are selected using light under a microscope Going through a fast and efficient photocuring process.

Using new DNA nanotechnology, the barcoded RNA sequences are then translated into coherent DNA strands, which can then be collected from the tissue sample and identified with the help from NGS. The Light-Seq process can be repeated with different barcodes for different cell populations in the same sample, which is left untouched for follow-up analysis. With performance comparable to single-cell sequencing methods, it greatly expands the depth and scope of possible investigations on a tissue sample. The method is published in Natural methods[BB1] .

“Light-Seq’s unique combination of features fills an unmet need: the ability to perform deep, image-based, spatially prescribed sequencing analysis of difficult or impossible to isolate cell populations. , or rare cell types in preserved tissues, with one-to-one correspondence of their highly refined gene expression state with spatial, morphological, and potentially disease-relevant features,” said Peng Yin, Ph.D. , one of four corresponding authors and a faculty member at the Wyss Institute, where his group developed Light-Seq, “so it has the potential to accelerate the process of biological discovery in various areas of biomedical research.” Yin is also a professor of systems biology at Harvard Medical School (HMS).

Bar code on the spot to 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 the Yin group at Wyss, and Emma West, Ph. D. in Constance Cepko’s laboratory at HMS. Previously, Kishi and Saka had developed SABER-FISH as a spatial transcriptomics method for imaging gene expression directly in intact tissues (on the spot). “With SABER-FISH, we were still far from capturing the complete gene expression programs of cells, with several thousand different RNA molecules per cell. RNA molecules are simply too dense to be captured in their entirety. using current imaging techniques,” said co-first and corresponding co-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 was a Wyss Technology Development Fellow on Yin’s team and is now pursuing a path to commercialization of Light-Seq with some of her co-authors.

“To specifically sequence cells in custom-selected locations of intact tissue samples, we developed a novel approach for photocrosslinking DNA barcodes to copies of RNA molecules, and a procedure based on the DNA nanotechnology that makes them readable by NGS, along with their attached RNA sequences,” said co-first author Liu, a postdoctoral fellow in Yin’s group who previously co-developed an encoding platform parallelized DNA bar graph for a super-resolution imaging method called “Action-PAINT” which has also become one of the core components of Light-Seq.

First, DNA primers “pair” with RNA molecules in cells and are extended to create copies of RNA sequences called complementary DNA sequences (cDNA). Then, the DNA barcode strands containing an ultrafast photocrosslinking nucleotide are in turn base-paired to the cDNAs in the cells. These become permanently bound when a target cell is illuminated under the microscope through a stencil-like optical device which keeps other non-target cells in the microscopic field dark and thus spares them from the reaction of photocrosslinking. After washing barcoded DNA sequences from cells that were not permanently bound on the spotthe procedure can be repeated with different barcodes and light patterns to label more regions of interest.

“To be able to integrate this barcoding workflow with NGS, we designed a new assembly reaction based on DNA nanotechnology. This innovation allows us to convert our barcoded cDNAs into contiguous read sequences We can then extract the full collection of barcodes bearing cDNA sequences from the sample and analyze them with standard NGS techniques,” explained Saka, one of the study’s corresponding authors who is currently lead. group at the European Molecular Biology Laboratory in Heidelberg, Germany. “Ultimately, each barcode traces the complete reading of the transcriptome back to the pre-selected cells in the tissue sample, which remain intact for further analysis. This gives us the unique chance to see the exact same cells again after sequencing for validation or further exploration.

Observing complex tissues and rare cells

Following Light-Seq’s first validation in cultured cells, Yin’s team wanted to apply it to complex tissue and partnered with the group of Constance Cepko, Ph.D. at HMS. Cepko is one of the study’s corresponding authors and the 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 forces with West in Cepko’s group to apply Light-Seq to cross-sections of the mouse retina and profile three main layers with different 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, tissue samples remained intact and could still be imaged for proteins and other biomolecules.

“By taking Light-Seq to the extreme, we were able to isolate the full transcriptome of a very rare cell type, known as ‘dopaminergic amacrine cells’ (DAC), which is extremely difficult to isolate due to its intricate connections with other cells in the retina, simply by picking up four to eight individual barcoded cells per cross-section,” West said. DACs are involved in regulating the circadian rhythm of the eye by adjusting the visual perception at different light exposures during the day-night cycle.” Light-Seq also detected RNAs specifically expressed in DACs at low levels, as well as dozens of DAC-specific RNA biomarkers that we know , had not been previously described, which opens up new opportunities to study this rare cell type. added West, who at the time of the study was a graduate student and then postdoctoral fellow at Cepko, and has now joined Kishi in his effort to commercialize Light-Seq.

Opening up the domain of spatial transcriptomics to NGS also adds information at the level of a single RNA species. “Our sequencing data clearly showed that Light-Seq can determine natural variations in RNA structure. In the future, we are very interested in using Light-Seq to better understand the interaction between the immune system , disease-spreading cells and different therapeutic strategies such as gene and cell therapy,” Kishi said.

“The Light-Seq technology developed in Peng Yin’s group as part of the Wyss Institute’s Molecular Robotics Initiative once again shows how pursuing a totally unconventional approach and harnessing the biology synthetic can lead to disruptive technology with great potential to advance both basic research and clinical medicine. said Wyss founding director Donald Ingber, MD, Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.

The other authors of the study are the members of the laboratory Yin Kuanwei Sheng, Jack Jordanides and Matthew Serrata. It was funded by the Wyss Institute through its Validation Project Program and Technology Development Fellowship at Kishi, Office of Naval Research Grants (Grant No. N00014-18-1-2549 ) and the National Institutes of Health (grant #2019-02433), as well as support from the European Molecular Biology Laboratory and the Howard Hughes Medical Institute.