DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

Abstract

Analyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in profiling the molecular constituents of cells, spatially mapping these constituents remains a disjointed and machinery-intensive process, relying on either light microscopy or direct physical registration and capture. Here, we demonstrate DNA microscopy, a new imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences, and infers physical images of the original transcripts at cellular resolution. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.

Intermediate states of molecular self-assembly from liquid-cell electron microscopy

“…The process contributes extra complexity to the heterogeneity in hybridization from the known varying mechanisms and kinetics. We did not further explore the issue at this time, but on physical grounds, we expect competition of this sort when DNA strands self-assemble in practically relevant technological situations of DNA profiling, origami, and DNA-controlled colloidal/ nanoparticle self-assembly (41)(42)(43). Three-strand experiments may shed some light on strand-displacement mechanisms, which are important in DNA nanotechnology.…”

A DNA nanoscope that identifies and precisely localizes over a hundred unique molecular features with nanometer accuracy

“…We wish to stress that each record molecule is a distance measurement, unlike the DNA microscope 9 that encodes distance non-linearly in the number of identical proximity-dependent records produced. Of course, not every measurement of the same distance produces a distance record of the same length, because the growing single-stranded recording primers are entropic springs and their growth process is stochastic.…”

“…However, they produce monochromatic images and can only discriminate molecular targets when they are resolved to near atomic precision, unachievable for many samples. At the other end of the spectrum, biochemical techniques like Hi-C can discriminate millions of DNA targets on chromosomes by sequencing them, however the contact densities currently obtainable from single nuclei Hi-C experiments preclude synthesizing this information into a structural model of the chromosome, while geometric models synthesized using data from ensemble Hi-C experiments have at best a local resolution of 5 kilobase pairs 6,7 . This fledgling, 'imaging-by-sequencing' field [8][9][10][11][12] has had two main experimental results. Our previous 'auto-cycling proximity recording' (APR) 8 effort demonstrated seven-point reconstructions (spaced ~30 nm apart) from simple, binary proximity data.…”

“…Our previous 'auto-cycling proximity recording' (APR) 8 effort demonstrated seven-point reconstructions (spaced ~30 nm apart) from simple, binary proximity data. The subsequent 'DNA microscope', a reaction-diffusion scheme, demonstrated thousands of single particle localizations but only ~10 µm resolution 9 . In contrast to these previous attempts, our DNA nanoscope leverages the particle homogeneity of DNA origami to produce a nanoscale-resolution spatial map of a hundred or more points by making thousands of independent, pairwise distance measurements.…”

A computational framework for DNA sequencing microscopy

“…At the time of publication, we are aware of concurrent works whose contributions are complementary to ours on development of DNA-sequencing-based microscopy (25,26). The former work experimentally demonstrates DNA microscopy with images of mRNA in cells using locally confined cDNA amplifications and polymerase extension-based fusion of barcodes to connect spatial patches.…”