A new spatial transcriptomics technology—the DNA nanotechnology-driven method called “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 through a photocrosslinking process.

With the help of DNA nanotechnology, the barcoded RNA sequences are translated into coherent DNA strands, which can then be collected from the tissue sample and identified using next-generation sequencing (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.

This work is published in Nature Methods, in the paper, “Light-Seq: Light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.”

“Light-Seq’s unique combination of features fills an unmet need: the ability to perform imaging-informed, spatially prescribed, deep-sequencing analysis of hard, if not 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, PhD, a core faculty member at the Wyss Institute.

To be able to specifically sequence the RNA molecules of neuronal cells in the functionally distinct layers of the mouse retina (graphic on the left), the team applied a new approach for “photocrosslinking” DNA barcodes to copies of RNA molecules in the retina’s individual light-selected layers (shown in magenta, light blue, and green). They then used a DNA nanotechnology-powered stitching technique to generate continuous sequences that could be mildly extracted from the tissue and eventually read via NGS. The cross-sections remained fully intact and were used for follow-up localizations of proteins encoded by RNA molecules that were enriched in the individual layers. [Wyss Institute at Harvard University]
Previously, Yin’s team had developed the spatial transcriptomics method SABER-FISH for imaging gene expression directly in intact tissues. “With SABER-FISH, we still were orders of magnitude away from capturing cells’ complete gene expression programs, with many thousands of different RNA molecules per cell. RNA molecules are just too densely packed to be captured in their entirety using present imaging techniques,” noted Jocelyn Kishi, PhD, a Wyss technology development fellow in the Yin lab. “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 advantages.”

“To specifically sequence the cells in custom-selected locations of intact tissue samples, we developed a new approach for photocrosslinking DNA barcodes to copies of RNA molecules, and a DNA nanotechnology-powered procedure that makes them and their attached RNA sequences readable by NGS,” said Ninning Liu, PhD, a postdoctoral fellow in Yin’s group.

First, DNA primers “base-pair” with RNA molecules in cells, and are extended to create cDNAs. Then, DNA barcode strands containing an ultrafast photocrosslinker nucleotide are base-paired to the cDNAs in the cells. These become permanently linked together when a target cell is lit up under the microscope through a stencil-like optical device that keeps other, nontarget cells in the microscopic field in the dark and thus spares them from the photocrosslinking reaction. After washing the barcoded DNA sequences out of cells that were not permanently linked in situ, the 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 engineered a new stitching reaction that is based on DNA nanotechnology. This innovation allows us to convert our barcoded cDNAs into contiguous readout sequences. We can then extract the complete collection of barcode-bearing cDNA sequences from the sample, and analyze them with standard NGS techniques,” explained Sinem Saka, PhD, currently a group leader at the European Molecular Biology Laboratory in Heidelberg, Germany and a former post-doc in Ying’s lab at the Wyss Institute. “Ultimately, each barcode traces the full transcriptome readout back to the pre-selected cells in the tissue sample, which remains intact for subsequent analyses. This provides us the unique chance to revisit the exact same cells after sequencing for validation or further exploration.”

Yin’s team applied Light-Seq to cross-sections of the mouse retina to profile three major layers with different functions. The researchers reached a sequence coverage comparable to single-cell sequencing methods and found that thousands of RNAs were enriched between the retina’s three major layers. They also showed that after sequence extraction, the tissue samples remained intact and could be further imaged for proteins and other biomolecules. Lastly, the team was able to isolate the full transcriptome of a dopaminergic amacrine cell of the retina—a rare cell type that is challenging to isolate.

“Our sequencing data clearly showed that Light-Seq can determine natural variations in the structure of RNAs. Going forward, we’re very interested in using Light-Seq to better understand the interplay between the immune system, disease-propagating cells, and different therapeutic strategies such as gene and cell therapy,” said Kishi, who is pursuing a path toward commercializing Light-Seq together with some of her co-authors.

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