“Think of moving this whole mountain up and down by, to scale, about a few meters,” he explains. “We measure how the resonance frequency of this mountain changes because it interacts with the ping pong ball. And all this we do without crushing the ping pong balls.”
Using this method, Hoogenboom and research associate Carl Leung were able to reproducibly pick out candy cane-like striations corresponding to the periodic major and minor groove spacing of DNA. Unlike standard amplitude-modulation AFM, the molecule was not squished in the process, producing a diameter of about 1.8 nm for double-stranded DNA (the accepted diameter is 2 nm). Even more remarkably, they not only detected the standard right-handed B-form DNA but also left-handed conformations in the same molecule, although the biological significance of that observation is unclear.
Now, Hoogenboom hopes to apply his technique to study DNA replication in real time. “It opens the possibility of doing experiments which help us to understand the replication process of genetic information at a scale which have been hardly accessible before, except by looking at just large ensembles of molecules,” he says.Regular or al dente?
Taekjip Ha also explores DNA at the single-molecule level. But rather than structure, Ha, a physicist at the University of Illinois, Urbana-Champaign, investigates the molecule's biophysical properties using a technique he has helped pioneer called single-molecule fluorescence-resonance energy transfer (FRET).
Ha's research interest concerns DNA flexibility. Over long distances, DNA can be thought of as a highly flexible rod — like cooked spaghetti. But there is controversy over the molecule's behavior over short distances, say 100 bases or so. One camp believes that DNA at this length scale behaves more like a rigid rod — dry spaghetti, perhaps. The other camp suspects the molecule at this length scale is more akin to a wet noodle. So, the question is, how does nature take its DNA: Regular or al dente?
This distinction is not merely academic. If DNA is rigid, then transcription factors must be able to recognize a linear molecule and bend it, a situation that requires energy. The alternative is to capture the transiently bent DNA form and stabilize it. “These are two extreme possibilities,” explains Ha.
To figure it out once and for all, Ha and his Ph.D. student Reza Vafabakhsh used single-molecule FRET and TIRF microscopy to measure the ability of molecules between 67 and 106 base pairs to form a “loop,” or circularize, joining two complementary single-stranded overhangs coupled to a FRET donor and acceptor pair. The molecules were tethered at their centers to a glass slide and monitored over time as salt was added to facilitate DNA hybridization.
The data showed that even molecules as short as 67-nucleotides can loop, albeit less efficiently than longer molecules, suggesting that transcription factors actually stabilize bent conformations rather than bend rigid ones. (Unpublished data push that limit down to 50 bases, says Ha.) Furthermore, they found that some sequences bend more efficiently than others, with equivalent-length molecules looping at different rates based on the number of consecutive adenine residues they contain.
According to Ha, these findings potentially explain nucleosome distribution across the human genome. “Sequence-dependent flexibility of the DNA may influence where the nucleosomes are formed on the DNA, and where it can also regulate the gene expression process,” he says.
Ha hopes to apply his technique to determine how modified bases, such methylcytosine, or mismatches influence flexibility. In the meantime, when it comes to the question of DNA flexibility, Ha says he's satisfied that question has been answered. “I think if someone wants to debate it further they'll have to come up with an even better experiment.”Hopping supercoils
Cees Dekker is applying extension of the technique Bustamante pioneered, magnetic tweezers, to explore DNA biophysics at longer length scales. Dekker is interested in the dynamics of DNA supercoils, hyper-wrapped topological structures resembling a tangled telephone cord that form, for instance, as molecular motors (such as RNA polymerases) travel along DNA templates.
According to Dekker, the management of supercoiling is a critical biological problem. “This coiled stuff is in your nucleus, and to express the genes there, to read off the genetic information, you have to locally uncoil [the] DNA molecule.”