Molecular Threading

Molecular Threading is a method for pulling single DNA molecules out of a droplet with the tip of a glass needle, holding them stretched in air between the needle and the surface of the liquid, and laying them straight on a dry surface. It was developed at Halcyon Molecular, a company that wanted to read DNA in an electron microscope, and was described in PLoS ONE in July 2013 by a group of authors that included Kent Kemmish and, as last author, George Church.^[1] The paper reported that threaded strands were straighter and could be packed far more closely than DNA stretched by molecular combing, the standard technique.

In solution a DNA molecule curls into a loose random coil, and a single strand coils more tightly still. Reading a molecule along its length, by fluorescence or in an electron microscope, needs it pulled straight and fixed to a surface. The paper traces mechanical stretching of DNA to a 1951 report in Nature, "Nucleic Acid: an Extensible Molecule?", in which fibres of the molecule's sodium salt were drawn out in air.^[2]

The usual method in 2013 was molecular combing, introduced in 1994. A drop of DNA solution is drawn across a treated surface, and the retreating edge of the liquid stretches molecules that have stuck to the surface by one end.^[3] In 1997 the technique was used to stretch DNA from the whole human genome for mapping.^[4] Combing needs the liquid to touch the surface where the DNA ends up, and its results depend on that surface and on the buffer. Optical tweezers, magnetic tweezers and the atomic force microscope can stretch one molecule at a time without a surface, but they cannot hand the molecule over to a substrate, and they are slow.^[1] For electron microscopy the DNA had to lie on films a few nanometers thick, fragile surfaces that suit wet methods poorly.

The needle is a pulled glass micropipette whose tip has been dipped in poly(methyl methacrylate) dissolved in acetone. The coating gives it, in the paper's words, "simultaneous DNA affinity and fluid hydrophobicity": DNA sticks to it and water does not. A computer-controlled piezoelectric stage moves the tip within a working volume 120 micrometers on a side, while separate positioners hold the droplet and the target surface and cameras watch from above and from the side.^[1]

The cycle has four steps. The tip pushes into a droplet of DNA solution, where one or more molecules bind to it. It withdraws, drawing the bound DNA out through the surface of the liquid. Surface tension at the meniscus pulls back on the part of the molecule still in the droplet, so the length suspended in air between droplet and tip is held taut, perpendicular to the liquid surface. Finally the tip touches the target, which takes the stretched DNA and wipes the tip clean for the next pass. A small sideways step between passes builds an array of parallel strands, and the cycle can run more than ten times a second.^[1]

The arrangement the authors preferred holds the droplet between two water-repellent surfaces, partly in contact with the target, so that the threads fan out from a band of combed DNA at the droplet's edge. The paper explains its name: "We call this configuration 'spider threading' since the threaded strands extend from a partially combed region like the legs of a spider."^[1] The stretching is reversible. A strand pulled past its limit slips out of the meniscus and relaxes while staying on the needle, and dipping the tip again restores the tension.

The method worked with single-stranded DNA made by rolling-circle amplification of the M13mp18 phage genome, about 380,000 bases long, and with the 167,000-base genome of bacteriophage T4 GT7, both pulled out to 40 micrometers. The 48,500-base genome of bacteriophage lambda, about 16 micrometers long when fully stretched, did not thread at that distance and could be drawn out only to less than 20 micrometers.^[1]

Strands were laid on soft silicone (PDMS), on silicon and silicon nitride membranes 5 nm thick, and, when neither droplet nor needle touched them, on floating carbon films as thin as 2.5 nm. Silicon films tended to stick to the needle or tear after a few hundred depositions; silicon nitride lasted longer.^[1]

The angles of 120 threads about 20 µm long fitted a normal distribution with a standard deviation of 1.16°. Against combing the paper reported these figures:

The paper also listed the method's faults. Some passes left nothing, some left two strands twisted together, and some strands forked. Threads were often thicker near the needle than near the droplet, a sign of hairpins, and the brightness of osmium labels showed that one thread could hold four times as many strands as another. Raising the DNA concentration above 25 ng/µL brought the yield of threads above 90 percent at the cost of more strands per thread.^[1]

Several of the authors gave Halcyon Molecular as their employer. The work was paid for by a grant from the United States Department of Energy and by the National Institutes of Health grant RC2 HG005592, the 2009 award that Kemmish had written for Church's laboratory and Halcyon.^[1][5] The paper was received on March 4, 2013, and published on July 31, 2013, about a year after Halcyon closed. Its section on future work proposes shielding the apparatus from "air currents, droplet evaporation, building vibration, and liquid currents in the droplet".^[1]

Kemmish's CV lists Molecular Threading among his contributions at Halcyon.^[5] The company he co-founded in 2018, Molecular Reality Corporation, works on reading single molecules as they pass through a hole in a membrane.

• Electron microscopy DNA sequencing
• Lambda phage DNA
• Molecular flossing
• Translocation (nanopore)