SABER tech gives a boost to the visualization of DNA and RNA

Researchers have used "FISH" analysis (in situ fluorescence hybridization) for decades to literally fish for specific sequences of DNA and RNA in intact cells and tissues in their vast seas of nucleic acid molecules. Because of its ability to microscopically illuminate specific sequences at the exact locations in which they reside, FISH has become a method of choice for the diagnosis of chromosomal abnormalities. It is the study of the three-dimensional organization of genomes in cells. nuclei, analysis of the immediate products of gene expression called messenger RNAs, etc.

However, it remains difficult to capture rare sequences with FISH, particularly in thick tissues that emit non-specific background fluorescence, and to detect multiple targets at the same time in a multiplexed analysis. Several methods have been developed to amplify weaker FISH signals, but they can not be easily customized, are unable to simultaneously visualize a large number of target DNA or RNA molecules, and are expensive and difficult use.

A collaborative research team from the Harvard Wyss Institute for Biologically Inspired Engineering and the Harvard Medical School (HMS) has developed "Signal Enhancement by Exchange Feedback" (SABER), a method Highly programmable and convenient that significantly improves sensitivity, customization and multiplexing. FISH analysis capabilities. The team demonstrated that SABER amplified FISH signals at distinct targets of RNA and DNA in the different layers of the mouse retina and simultaneously visualized up to 17 different target regions on the human X chromosome. In addition, they used SABER as an effective tool to identify a genomic element controlling transcription of a specific gene expressed in a particular type of retinal neuron. Their study is published in Nature Methods.

"With SABER amplified FISH analysis and its sensitivity, multiplexing potential, practicality and cost-effectiveness, we have created a way to overcome the main limitations of existing methods and provide researchers more powerful from a wide range of analyzes, ranging from basic research, to the discovery of biomarkers and to the development of therapies, "said Peng Yin, Ph.D., senior faculty member of the Wyss Institute, which corresponded to the study jointly with Constance Cepko, Ph.D., Bullard Chair of Genetics and Neuroscience of the Blavatnik Institute at HMS and Brian Beliveau, Ph.D. , former Damon Runyon Postdoctoral Fellow in Yin Group and now Assistant Professor in the Department of Genome Sciences at the University of Washington in Seattle.

Yin is also co-director of the Wyss Institute's Molecular Robotics Initiative and Professor of Systems Biology at Harvard Medical School; and Cepko is also a researcher at the Howard Hughes Medical Institute and a member of the Harvard Stem Cell Institute.

In SABER, the researchers first programmed the "PRimer Exchange Reaction" method (PER) previously described by the Yin group to synthesize a longer concatemer of identical shorter sequences using a hairpin structure. DNA hair folds automatically. This modified mechanism has a similarity to the natural enzymatic mechanism that extends "telomeres" to the ends of chromosomes with identical repetitions of DNA to protect them from degradation, but can be run in a test tube.

The PER-generated concatemers are then hybridized via short sequences of complementary handles with their target DNA or RNA sequences into fixed cells and tissues where they provide scaffolding with multiple binding sites for short fluorescent oligonucleotides ("imagers") added thereafter. "Unlike conventional FISH analysis, SABER not only allows us to link one, but more than one fluorescent dye molecule to a single nucleic acid target, which can dramatically increase the signal strength from its location." in the cell, and we can even Jocelyn Kishi, Ph.D., co-first author of the study and postdoctoral fellow working with Yin, added, "Improve it by creating branched structures by multiplying the hybridization steps , with additional concatemers developed from internal branching points: SABER at Beliveau "This opens up a wealth of new opportunities."

"SABER allows us to adjust the signal strength to the abundance and location of specific RNA and DNA targets in FISH analysis and, as the concatenators of multiple targets can be synthesized en bloc at the same time. In advance, SABER provides us with standardized sets of probes that can be used – easily generated and reused in different studies, "said Béliveau. "In addition, by repeatedly cleaning complementary imagers of a set of targets of a sample and replacing them with imagers binding to other targets in a process called exchange of information. DNA, we were able to further multiplex our approach. "

The spectral characteristics of distinguishable fluorescent dyes allow researchers to analyze up to four targets at the same time using conventional fluorescence microscopy. With DNA-Exchange, a set of imagers can be removed from one sample and replaced by another linking to PER-generated hubs at different target sites, and the process can be repeated multiple times. Using the multiplexing potential of "Exchange-SABER", the team visualized 17 distinct regions of the human X chromosome in one shot using the same type of microscopic equipment.

Applying SABER to thick tissue, Constance Cepko's group at HMS demonstrated the performance and utility of the retinal method, which is layered with a wide variety of neuronal cell types. "We had previously applied unicellular sequencing to dissociated retinal tissue, but we were unable to use the markers obtained to simultaneously identify multiple cell types in intact tissue," said co-lead author, Sylvain Lapan, Ph.D. .D., Postdoctoral fellow in Cepko's group. "By providing multiplexed detection of marker transcripts, SABER allows us to close this loop and add a spatial dimension to our analyzes."

Emma West, co-first author, has developed for their analysis a method that, based on fluorescence staining on the surface of cells, allows to computationally map all the cells contained in a cross section of the retina to positions specific. This allowed the team to assign individual signals obtained by SABER-enhanced FISH analysis to exact 3D spaces occupied by cells. "By modeling cell coordinates and transcription positions, we can create a quantitative representation of our tissue that is quantitative at the cell level." This is useful not only for identifying cell types, but also for analyzing cell types. phenotypes and the activity of introduced genetic elements, "said Emma West, co-first author, who is a graduate student in the Cepko group.

In addition, the team used SABER-FISH as an effective tool for identifying genomic elements, known as enhancers, that stimulate gene expression in retinal cell types. specific. "We have introduced into the retina recombinant DNA constructs in which these elements were individually coupled to a reporter by performing a SABER-FISH analysis to quantify the number of copies of constructs in single cells, as well as the number of copies. 39, reporter's expression, we have been able to correlate the activity of a specific activator with the specific expression of the cell type, "said Lapan.

"The simpler and cheaper sensitivity and workflow of SABER-FISH allows us to collect much more information from experiments in which we manipulate the retina to understand the regulatory networks that regulate it." development, and evaluate the effectiveness of our gene therapy approaches for eye diseases, "Cepko said. "It is important to note that this approach can also improve research on many other types of tissues and organs."