DNA microscopy
The CRISPR/Cas pioneer has created a new microscopy method. You don't need a microscope for it
Instead of light, he uses DNA molecules, and instead of eyes, he uses a sequencer and a computer
Polina Loseva, "The Attic"
American scientists have come up with a new way to see the location of RNA molecules in a cell. They marked strands of different RNAs distributed throughout the cell with DNA molecules, and then sequenced its genome to then build an image based on these data. The resulting images are not inferior in detail to the results of light microscopy, and also open up new opportunities for scientists: previously, they could not see such a detailed image of the "genetic insides" of a cell at a time.
On the right is a photo of cells producing red and green fluorescent proteins in a conventional microscope. On the left is the result of DNA microscopy. Red and green are fluorescent proteins, gray is actin, white is a protein involved in the breakdown of glucose. The size of the ruler is 100 microns. A drawing from an article by Weinstein et al. DNA Microscopy: Optics-free Spatio-genetic Imaging by a Stand-Alone Chemical Reaction.
Modern microscopy methods are good at determining the shape of cells and the density of the molecules inside them, but they do not allow recognizing individual molecules. You can, of course, introduce special dyes or glowing probes into the cell, but for each molecule of interest to scientists, you need your own probe, preferably not overlapping in the radiation spectrum with others. These technologies work for individual proteins, but become inconvenient when it comes to nucleic acids, for example, RNA molecules.
RNAs are copies of DNA, "extracts" from genes, looking at which the cell synthesizes proteins. Unlike DNA, which is concentrated in the nucleus, RNAs are distributed throughout the cytoplasm, and sometimes are grouped in the place where the proteins they encode are needed. To consider the location of RNA molecules in a cell, the group of Zhang Feng (the man who first made CRISPR/Cas work in human cells) from the Massachusetts Institute of Technology created a method DNA microscopy. However, it has little in common with the microscopy we are used to: for example, it does not need a microscope, but a sequencer and a special algorithm that builds a picture based on sequencing data.
To begin with, the cells we are interested in fix and make small holes in the membrane. Polymerases, enzymes for copying nucleic acids and DNA probes are injected into these holes: DNA sequences that are complementary (that is, capable of sticking) to the RNA molecules under study and carry unique labels at the end.
As a reference point – relatively speaking, the "zero coordinate" – scientists used the RNA of the protein actin. There are a lot of these RNAs in various parts of the cell, so it was logical to expect that they would be able to reach any point of the cell.
The probes injected into the cell stick to all the RNAs, and the cell completes the probes to the end – for this, in fact, it requires polymerases. Strands are obtained in which the RNA sequence is followed by a unique label. Then primers are thrown into the cells – a set of short, unique pieces of DNA. They stick to the ends of the threads, the cage also completes them to the end. The resulting molecules consist of an RNA sequence, a unique label and a primer.
Then these molecules stick together with their ends, since the primers are complementary. The result is a long thread: "zero" RNA, unique label 1, fused primers, target RNA, unique label 2. The closer the "zero" RNA and target RNA are located in the cell, the more such strands will turn out, and, accordingly, the distance between the two can be estimated by their number. fragments of RNA.
Finally, the researchers sequence the nucleic strands from the cell and evaluate their concentration. According to the ratio of the couplings of "zero" RNAs with different RNA targets, a computer algorithm restores their distribution throughout the cell and even its shape.
In the future, researchers hope to study how RNAs are distributed in nerve and immune cells (for which it is important to interact with other cells in their different parts), as well as to identify unique RNA sequences with point mutations in immune and cancer cells.
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