Point the spotlight at the cells in your tissue so RNA can tell their story!

Under the microscope, researchers often observe different types of cells organizing themselves in tissues in unique patterns, or sometimes a rare cell type that stands out
by occupying unique locations, displaying unusual shapes, or expressing specific biomarker molecules.
To determine the deeper significance of their observations, they developed a method to access the cell's gene expression patterns (transcriptome) by analyzing gene-derived RNA molecules present in the cell, which they could match
to the cell's shape, spatial location, and molecular biomarkers.

However, these "spatial transcriptomics" methods still capture only a fraction of the total RNA molecules of cells and do not provide the depth and quality analysis offered by single-cell sequencing methods, developed to study the transcriptome of individual cells isolated from tissues or biological fluids through next-generation sequencing (NGS) technology
.
They also don't allow researchers to lock down specific cells based solely on their location in tissues, which would greatly facilitate the study of isolated cell populations, or rare, hard-to-isolate cells such as rare brain cells with unique functions, or immune cells
that invade tumors.
In addition, many spatial transcriptomics and all single-cell sequencing methods make it impossible for researchers to revisit samples for subsequent analysis due to disruption of the original tissue environment, and these methods require specialized instruments or reagents, making them costly
.

A new advance in Harvard University's Weiss Institute for Biostimulation Engineering overcomes these limitations
with a DNA nanotechnology-driven approach called "Light-seq.
" Light-Seq allows researchers to "geotag" entire RNA sequences with unique DNA barcodes unique to a small number of cells of interest
.
These target cells are selected
under the microscope through a fast and efficient photocrosslinking process.

With the help of a new DNA nanotechnology, barcoded RNA sequences are translated into coherent DNA strands, which can then be collected from tissue samples and identified
using NGS.
The Light-Seq process can be repeated with different barcodes for different cell populations in the same sample and left as is for subsequent analysis
.
Its performance is comparable to single-cell sequencing methods, greatly broadening the depth and scope
of tissue sample investigation.

Dr.
Peng Yin, one of the corresponding authors and a core member of the Wise Institute, said: "Light-Seq's unique combination of capabilities fills an unmet need: the ability to perform image information, spatial conditioning, deep sequencing analysis of hard-to-isolate cell populations or rare cell types in preserved tissues, one-to-one correspondence with their highly refined gene expression states and spatial, morphological, and underlying disease-related characteristics, and therefore has the potential to rapidly advance the biodiscovery process in biomedical research
。 ”

From in situ barcoding to ectopic sequencing

The Light-Seq project was spearheaded by Dr.
Jocelyn (Josie) Kishi, Dr.
Sinem Saka and Dr.
Ninning Liu in Yin's group and Dr.
Emma West in Constance Cepko's lab at HMS
.

Prior to this, Kishi and Saka had developed SABER-FISH as a spatial transcriptomics method to image
gene expression directly in intact tissue (in situ).
"With SABER-FISH, we are still orders of magnitude away from capturing the complete gene expression program in cells, each with thousands of different RNA molecules
.
RNA molecules are too dense to capture them intact with current imaging techniques," said
co-first author and co-corresponding author Kishi.
"Light-Seq solved this problem by combining high-resolution barcode labeling with whole transcriptome sequencing via NGS, giving us the best results of both worlds and the additional key advantage
.
" At the time of the study, Kishi was a Wyss technology development researcher on Yin's team and is now pursuing a path to commercialization of Light-Seq with some of her co-authors
.

"To perform specific sequencing of cells at custom-selected locations of intact tissue samples, we developed a new method to crosslink DNA barcoding light onto copies of RNA molecules, as well as a DNA nanotechnology-driven program that makes them and their attached RNA sequences readable by NGS
.
" Co-first author Liu, a postdoc on Yin's team, previously co-developed a parallel DNA barcoding platform for a super-resolution imaging method called Action-PAINT, which has also become one of the core components of
Light-Seq.

First, DNA primers are "base-paired" with RNA molecules in cells and expanded to create copies
of RNA sequences called complementary DNA sequences (cdna).
The DNA barcode strands containing ultrafast photocrosslinker nucleotides are then base-paired
with the cdDNA in the cell.
When target cells are illuminated under the microscope through a template-like optical device that places other non-target cells under the microscope in the dark, thus shielding them from the photocrosslinking
reaction.
After washing away barcoded DNA sequences in cells that are not permanently connected, the process can be repeated with different barcode and light patterns to mark more regions of
interest.

"To be able to integrate this barcode workflow with NGS, we designed a new splicing reaction
based on DNA nanotechnology.
This innovation allows us to convert our barcode cdDNA into a continuous readout sequence
.
We can then extract the complete set of cDNA sequences containing barcodes from the sample and analyze them with standard NGS techniques, and ultimately, each barcode traces the complete transcriptome readout back to pre-selected cells in the tissue sample, which remain intact for subsequent analysis
.
This gives us a unique opportunity to revisit the exact same cells
after sequencing validation or further exploration.
" ”

Observe complex tissues and rare cells

With Light-Seq being first validated in cultured cells, Yin's team wanted to apply it to complex tissues and work with the Constance Cepko team
at HMS.
Cepko is one of the corresponding authors of the study, which studied the development of
the retina as a model of the nervous system.
Kishi, Saka, and Liu collaborated with West from Cepko's team to apply Light-Seq to a cross-section of the mouse retina and analyze 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 remain intact and can be further imaged for proteins and other biomolecules
.

"Taking Light-Seq to the extreme, we were able to isolate the complete transcriptome of a very rare cell type, known as 'dopaminergic non-secretory cells' (DACs), which are difficult to isolate due to its complex connections to other cells in the retina, with only 4 to 8 independent barcoded cells
extracted per cross-section," West said.
DACs are involved in regulating the circadian rhythm of the eye by fine-tuning the visual perception
of different lights during the circadian cycle.
West added, "Light-Seq also detected RNA specifically expressed in DAC at low levels, as well as dozens of DNA-specific biomarkers, which, to our knowledge, opens up new opportunities
to study this rare cell type.
" West was a graduate student at Cepko when he conducted the research, then a postdoc, and has now joined Kishi's Light-Seq commercialization efforts
.

Opening the field of spatial transcriptomics to NGS also increases information
at the individual RNA species level.
"Our sequencing data clearly shows that Light-Seq can identify natural changes in
RNA structure.
Going forward, we are very interested
in using Light-Seq to better understand the interactions between the immune system, disease-transmitting cells, and different therapeutic strategies such as gene and cell therapy.
Kishi said
.

"The Light-Seq technology, developed by Peng Yin's team in the Molecular Robotics Program at the Wyss Institute, once again shows that pursuing a completely unconventional approach and harnessing synthetic biology can lead to a disruptive technology with great potential to advance basic research and clinical medicine
.
"