Unusual Structures in Super-Long CAG Repeat DNA Figure 7. Convoluted structures resist considerable pulling force. (A) AFM image of a DNA molecule containing 360 CAG repeats (total length 1211 bp) showing a streptavidin molecule, attached to a terminal biotin moiety. Scale bar, 100 nm. (B) AFM image of a 272-repeat DNA molecule showing an anti-DIG antibody molecule, attached to a terminal DIG moiety. Scale bar, 100 nm. (C) Experimental rationale. A silicon nitride cantilever and a mica disk were both amino-functionalized by incubation with APTES. Glutaraldehyde was then used to couple streptavidin to the cantilever and anti-DIG to the mica. DNA fragments were bound to the steptavidin via a biotin tag on the DNA. The DNA was lowered onto the antibody-coated mica to allow the DIG tag on the DNA to bind to its antibody on the mica. The cantilever was then retracted, and the forces applied during the retraction are measured. Two possible outcomes are depicted: (1) the convoluted DNA is unwound; (2) the DNA remains convoluted and the antigen-antibody bond is ruptured. (D) Typical force curve for a DNA fragment containing 114 CAG repeats, showing ‘approach’ (blue) and ‘retract’ (red) traces. Note the multiple force peaks, the final rupture distance (41 nm) and force (200 pN). 14/150 retractions (9%) resulted in a measurable force peak. (E) Typical force curve for a DNA fragment containing 360 CAG repeats. Note the multiple force peaks, the final rupture distance (117 nm) and force (159 pN). 22/124 retractions (18%) resulted in a measurable force peak. (F) Force curve for a 360-repeat fragment showing a very long rupture distance (260 nm) and a large force (529 pN). doi:10.1371/journal.pone.0017119.g007 In the example shown (Figure 7F), the pulling distance was 260 nm and the pulling force was 529 pN. These long pulls likely represent the behaviour of unconvoluted DNA present in the long- repeat sample. Overall, these pulling experiments demonstrate that the antigen-antibody bond is ruptured before the DNA convolutions are unwound, supporting the idea that the convo- luted DNA contains stable structures such as hairpin loops. Discussion AFM allows high-resolution, single-molecule imaging of DNA with minimal sample preparation. We have shown previously that this imaging method provides structural information not attainable using other methods, such as EM. In this study we have used AFM imaging of DNA containing super-long CAG repeats to reveal the presence of various unusual non-B-form structures, including hairpin loops and large convoluted structures. By contrast, in previous EM-based studies, the observed features consisted almost exclusively of short spurs and kinks [22,23]. The unusual structures reported here are consistent with both the prediction [42] and experimental evidence [43] that DNA composed of long stretches of CAG repeats is particularly flexible. Importantly, we also show that the prevalence of these anomalous structures increases with increasing CAG repeat length. Maintained sensitivity to the restriction enzyme TseI rules out the presence of extensive regions of single-stranded DNA. On the other hand, sensitivity to digestion by mung bean nuclease is consistent with the presence of hairpin loops. The fact that forces sufficient to rupture antigen-antibody bonds fail to unwind the convoluted DNA indicates the presence of stable unusual DNA structures. It is known that a significant change in the phenotype of the R6/2 mouse occurs at a repeat length of around 300 CAGs [17,18]. Specifically, increases in repeat length have been shown to cause a delayed onset of disease and prolonged survival, from PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e17119