Decades of crystallographic and NMR studies have produced canonical structural models of short DNA. However, no experimental method so far has been able to test these models in vivo, where DNA is long and constrained by interactions with membranes, proteins, and other molecules. Here, we employ high-resolution frequency-modulation AFM to image single long poly(dA)-poly(dT), poly(dG)-poly(dC), and lambda DNA molecules interacting with an underlying substrate that emulates the effect of biological constraints on molecular structure. We find systematic sequence-dependent variations in groove dimensions, indicating that the structure of DNA subject to realistic interactions may differ profoundly from canonical models. These findings highlight the value of AFM as a unique, single molecule characterization tool.
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