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Researchers open the door into a new branch of cellular analysis

Written by Aaron Khemchandani, Science Writer.

New advances in DNA nanotechnology have allowed scientists to significantly improve the depth of investigations possible on tissue samples, paving the way for progress in fundamental research and clinical medicine.

Overcoming challenging limitations

Despite recent progress, existing spatial transcriptomics approaches are still only able to capture a fraction of a cell’s total RNA molecules, and cannot provide the quality of analysis delivered by single-cell sequencing methods. They also do not allow researchers to selectively examine specific cells based on their location within a tissue, which would facilitate the pursuit of rare, difficult-to-isolate populations.

However, researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University have developed a DNA nanotechnology-based technique entitled ‘Light-Seq’. The paper, published in the journal Nature Methods, details how Light-Seq allows scientists to geographically tag RNA sequences with individual DNA barcodes exclusive to a small number of cells. They also go on to discuss how these cells are selected using a process known as photocrosslinking. The potential importance of this new approach for future spatial transcriptomics research cannot be understated.

Isolating the impossible

The high selectivity of Light-Seq would provide scientists with the ability to conduct deep, detailed genomic analyses of cells previously considered to be almost “impossible to isolate,” according to corresponding author and Core Faculty Member at the Wyss Institute, Peng Yin, PhD.

“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,” he said. “It thus has potential to fast-forward the biological discovery process in various biomedical research areas.” 

Figure 1. An overview of Light-Seq (ROI = region of interest, NGS = next-generation sequencing, IF = immunofluorescence). Credit: Kishi et al., 2022.

First, DNA primers base-pair with RNA molecules within cells, and are then extended to generate DNA sequences complementary to the RNA molecules (cDNA). After this, DNA barcode strands containing a “photocrosslinker nucleotide” are base-paired to the aforementioned cDNA sequences. The photocrosslinker nucleotide is named as such because once a target cell is lit up, the DNA barcode strands and cDNA molecules become permanently linked. A stencil-like optical device is then used in conjunction with the microscope to keep other, non-target cells in the field dark, thus sparing them from the photocrosslinking reaction which is triggered by light. This allows researchers to selectively target a very narrow range of cells.

A tool of unlimited potential

After validating the use of Light-Seq in cultured cells, Yin’s team partnered with Harvard Medical School (HMS) to apply it to a complex tissue. The joint effort allowed researchers to apply the technique to cross-sections of the mouse retina and profile three major layers, each of which had different functions.

“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’ (DACs), which is extremely hard to isolate because of its intricate connections to other cells in the retina, by retrieving merely four to eight individually barcoded cells per cross-section,” said Emma West, a scientist at HMS. DACs play an important role in regulating the eye’s circadian rhythm by adapting visual perception to various light exposures during the regular day-night cycle.

“Light-Seq also picked up RNAs that were specifically expressed in DACs at low levels as well as dozens of DAC-specific biomarker RNAs that, to our knowledge, had not been described before, which opens new opportunities to study this rare cell type,” she added.

Novel windows of opportunity

Light-Seq technology highlights significant advancements in the field of spatial transcriptomics. The possibilities for scientists are seemingly endless, as the approach opens up countless opportunities to study a host of extremely rare cells. While the subject of DNA nanotechnology is relatively new among researchers, the establishment of this technique could well provide a suitable springboard from which this field can be elevated to new heights.