Even though nearly 60 years have passed since James D. Watson and Francis Crick solved the structure of DNA, we still have much to learn about the iconic double helix.
“These are complex molecules, and only now are [we] developing the techniques, the technology that allows us to study them systematically with high precision,” says Carlos Bustamante, professor of molecular and cell biology at the University of California, Berkeley.
Bustamante should know: In 1992, his lab pioneered single-molecule biophysical studies of DNA when they attached a magnetic bead to the double helix and measured elasticity as force was applied along the molecule's helical axis. Yet 13 years later, using the same technique, they were surprised to learn that the textbook understanding of how DNA responds to such force — an understanding that they, in part, developed — was incorrect.
The conventional wisdom, explains Bustamante, was that pulling the DNA molecule taut would cause it to unwind and then stretch. And indeed, in 1996, his team determined that by yanking on a piece of DNA with progressively greater force that the molecule can exist in any of three elasticity “regimes.” But, in 2005, his team discovered that during the first regime, as the molecule is being extrended, it actually overwinds instead of unwinding. It is only at a certain force level that the molecule begins to unwind, and finally, to physically stretch. The idea that a helix would overwind upon being extended is clearly counterintuitive. And yet, this is precisely what was observed. How a helix responds (unwinding or overwinding) to extension is determined by its so-called torsional-extension coupling constant. That constant was believed to hover around +200 pN nm. But, in fact, Bustamante's team measured it at -90 pN nm.
“We discovered that not only did we have the wrong value for the torsional-extension coupling constant of DNA, but we actually had the wrong sign for it,” says Bustamante. “So, there are still a lot of things that need to be understood about a molecule like DNA.”
Call it a technology tipping point in DNA structure research, but today scientists are applying a battery of novel microscopy techniques to understand DNA at length scales from the atomic to the genomic. That's important, says Bustamante, because each approach reveals a different aspect of the molecule, as in the classic story of three blind men describing an elephant's appearance. “It is only through the concerted and coordinated view of the molecule from different aspects that we are going to ultimately get a much better appreciation of what makes the DNA such a special molecule.”
Single-molecule candy canes
Bart Hoogenboom takes his view of DNA from atomic force microscopy (AFM).
In AFM, a sharp microscopic tip at the end of a flexible diving board-like cantilever scans over the sample line by line like a Gramophone needle, mapping the topography of the sample beneath it. Typically, that mapping is achieved by measuring the deflection of the cantilever as the stylus is dragged across the sample, but that scanning mode can both drag and distort the molecules being imaged. As a result, DNA in an AFM typically lacks the resolution of crystalline samples, looking more like a squashed rope with its helical substructure obscured.
Yet AFM, explains Hoogenboom, a University College London physicist, offers multiple advantages for imaging DNA. Unlike optical microscopy, AFM's resolution is not limited by the diffraction limit of light (200 nm or so); it offers single-digit nanometer resolution. And unlike electron microscopy and X-ray diffraction, AFM can be applied to individual molecules in aqueous solution.
Hoogenboom and his team recently optimized the method by implementing a suite of changes, including shrinking the cantilever about 10 times and measuring topography by changes in the cantilever resonance frequency as it passes over the sample. They call the method “constant-amplitude phase-modulation AFM.”
Imagine scaling up the atoms in the sample to the size of a ping pong ball, explains Hoogenboom. The cantilever tip would be like an inverted mountain, several miles high, hovering above it.