height of 0.4 nm, which is typical of the height of double-stranded (ds) DNA, as determined by AFM imaging (e.g. [26]; Figure 4A). (The width of the DNA is considerably overestimated because of the geometry of the scanning tip, so this parameter does not yield useful information). By contrast, the height of a typical ‘protrusion’ (0.7 nm) is almost twice that of normal dsDNA (Figure 4B). This structure might therefore represent self-associating hairpins. The heights of the spurs in the convoluted DNA (Figure 4C) are close to that of normal dsDNA, suggesting that these structures might be single hairpins. If hairpin loops do exist within the convoluted DNA, then they will contain both short single-stranded regions at their tips, and mismatched bases. Both of these features should be susceptible to cleavage by mung bean nuclease [21,22]. When DNA containing 360 CAG repeats was incubated with mung bean nuclease without subsequent melting, it appeared similar to undigested DNA, although there was an increase in the number of short fragments (Figure 5A). However, if the DNA was melted after the reaction and then allowed to reanneal, very little full-length linear DNA remained, and instead many smaller fragments were seen (Figure 5B). These data best fit a scenario in which mung bean nuclease cleaves mismatches in hairpins - generating some short fragments before melting - and also short ssDNA in hairpin loops, resulting first in nicks, and then in many very short fragments upon DNA melting. These results indicate that hairpin loops are indeed present in the convoluted DNA, and that even apparently ‘normal’ linear DNA contains short hairpins below the resolution limit of AFM. The Unusual Structures are Sensitive to Cleavage by the Type-III Restriction-Modification Enzyme EcoP15I The sequence 59-CAGCAG-39 is the recognition sequence for the Type III restriction-modification enzyme EcoP15I [37,38]. For efficient cleavage, EcoP15I normally requires the interaction of two enzyme complexes that bind to sites oriented in a head-to- head fashion. One enzyme translocates towards the other, bound at its target site, and cleavage occurs upon collision. It has been shown previously that EcoP15I cuts CAG repeat DNA, provided that a reverse recognition site is present in the plasmid [39], and it was demonstrated that this enzyme could be used to count the number of CAG repeats in a DNA sequence. We decided to test whether EcoP15I would selectively cut convoluted DNA, even though the DNA does not contain a reverse recognition sequence, Unusual Structures in Super-Long CAG Repeat DNA reasoning that the geometry of the convolutions might allow collision between two enzyme complexes even in the absence of a reverse recognition sequence. We found that EcoP15I treatment did not cleave linear DNA containing a relatively short (216) CAG repeat (compare Figure 6A with Figure 6B). Interestingly, the treated molecules often bore blobs (arrows in Figure 6B), which are likely to be enzyme complexes that have stuck to the ends of the DNA. In contrast, EcoP15I treatment of DNA containing 360 CAG repeats led to the generation of shorter linear fragments (compare Figure 6C with Figure 6D), many of which again bore blobs at their ends (arrows in Figure 6D), small, tangled structures (arrowheads in Figure 6D), and normal full-length linear DNA. The frequency distribution of lengths for the linear subpopulation in the untreated sample (Figure 6E) shows a single peak, and the mean length was 370626 nm (n = 50), as reported above. The corresponding distribution for linear DNA in the EcoP15I- digested sample (Figure 6F) has two peaks, one at 372629 nm, and a broader peak, encompassing fragments with blobs at their ends, at 141668 nm (n = 50). EcoP15I treatment also caused a fall in the volume of the convoluted structures from 9106330 nm3 to 3 3806190 nm . These results indicate that EcoP15I does indeed preferentially digest the convoluted DNA, leaving behind a mixture of short linear fragments of various lengths and tightly- wound tangles. The Unusual Structures are Very Resistant to Unfolding The results presented so far suggest that the convoluted DNA is not loosely coiled, but rather contains non-B-form structures. To test this idea further, we attempted to measure the force required to ‘untangle’ the convoluted DNA. We reasoned that loosely coiled DNA should be untangled at a relatively low force, but that hairpin loops, for example, should resist large pulling forces. We produced DNA containing either 114 or 360 CAG repeats, each bearing a biotin tag at one end (where the forward primer anneals) and a digoxigenin (DIG) tag at the other end (where the reverse primer anneals). AFM images showed that the biotin tag could be decorated with a streptavidin molecule (Figure 7A), and the DIG tag could be decorated with an anti-DIG antibody (Figure 7B). For single-molecule force spectroscopy, the AFM cantilever was derivatized with streptavidin, and the mica surface was coated with covalently bound anti-DIG antibody (Figure 7C). DNA was attached to the streptavidin-derivatized cantilever via the biotin Figure 5. Effect of mung bean nuclease indicates the presence of hairpins in the convoluted molecules. (A) DNA containing 360 CAG repeats that had been treated with mung bean nuclease. Note the presence of an unusual number of short fragments. (B) The same DNA that had been treated with mung bean nuclease, and then melted and reannealed. Note the almost complete absence of full-length linear DNA. Scale bars, 250 nm. doi:10.1371/journal.pone.0017119.g005 PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17119