Unusual Structures in Super-Long CAG Repeat DNA receptor DNA that was exclusively double-stranded was also efficiently cleaved by TseI, but in this case there was no residual ssDNA (Figure S2B). These results demonstrate that TseI cuts dsDNA that contains the appropriate target sequence, but not the corresponding ssDNA. An AFM image of DNA containing 585 CAG repeats showed the usual pattern of normal and unusual structures (Figure 3C). An extensive digestion of this sample with TseI at 80uC resulted in the removal of all conformations of DNA (Figure 3D), showing that it was predominantly double stranded. A dense homogenous ‘background’ of short (,6-bp) fragments remained, together with a few aggregates. One fragment of ,60 bp, which likely represents a flanking region, can also be seen. Interestingly, when a limited TseI digestion was carried out at room temperature, the enzyme preferentially cleaved linear regions of DNA, leaving behind the contorted regions (Figure 3E, F). These results rule out the presence of extensive regions of ssDNA in the unusual structures, and also indicate that the convolutions sterically hinder access of the restriction enzyme to the DNA. The Unusual DNA Structures Include Hairpin Loops To probe the nature of the unusual DNA structures further, we took cross-sections through them and compared the profiles to those of ‘normal’ DNA. A section through linear DNA indicates a Figure 4. Sections through DNA molecules showing either normal or unusual structures. (A) Normal DNA (216 CAG repeats). Scale bar, 100 nm. (B) Protruding DNA (360 CAG repeats). The cross-section shows a width comparable to that of normal dsDNA but a greater height, suggesting an imperfect hairpin or hairpin-like structure. Scale bar, 100 nm. (C) Convoluted DNA (585 CAG repeats). The cross-section indicates protrusions similar in height and width to dsDNA. Scale bars, 50 nm. doi:10.1371/journal.pone.0017119.g004 PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17119