DNA Shape refers to physical properties of DNA duplex (i.e., flexibility, bendability, electrostatics). It has long been recognized that DNA Shape dictates specific DNA recognition beyond the linear base sequence. However, DNA shape is collectively determined by local core sequence, peripheral elements, and other factors (e.g. topology). While we have the capability of providing each individual the linear sequence of their DNA genome, we have not yet able to deduce, a prior, DNA shape information from a given linear genome sequence.

Using the R5-family of nitroxide labels that allow one to scan a DNA duplex, we have demonstrated a number of approaches for deducing free DNA shape and to monitor conformation of DNA duplexes bound by proteins or small molecules. With our methods, we extract local structural and dynamic information by analyzing X-band continuous-wave (cw-) EPR spectrum that reports on rotational motion of singly-labeled nitroxides (read more…). The detailed spectral lineshape analysis is combined with global R5 scanning to yield a map of the local DNA environment along the nucleotide positions (read more…). In free duplexes, the R5 maps reflect sequence-dependent variations of helical parameters (e.g., roll) and reveal shape differences between different DNAs (read more…). Furthermore, we have developed an integrated approach to deduce atomistic models of free DNA duplexes using the R5 tool-kit (read more…). The method measures a large number (15-20) of inter-R5 distances for a given DNA duplex, and applies these experimental constraints to a large pool (~10,000) of models to deduce the target structure. These SDSL methods have been applied to analyze the p53 response elements (p53RE), a family of DNA sequences specifically recognized by the p53 tumor suppressor, a transcription factor with various essential roles in maintaining the integrity of the human genome. Our work on two p53REs indicates that the intrinsic DNA shape differences are exploited by the protein, resulting in bound complexes with different modes of DNA deformation yet maintaining all the protein-protein and protein-DNA contacts (read more…). The work established a direct connection between DNA sequence-dependent shape and deformation upon p53 protein binding, which may contribute to specificity in p53 recognition.

Local DNA Physical Environment at Individual Base-Pairs R5-Tool-Kit Identifies DNA-Shape Dependent Binding Modes

Our current projects continue to pursue in-depth understanding on how DNA shape impacts target discrimination by CRISPR-Cas9 and Cas12a. Cas9 and Cas12a recognize DNA targets via a series of RNA/DNA directed conformational changes. The key steps to establishing the correct target, including PAM recognition, PAM-adjacent DNA distortion for R-loop initiation, DNA unwinding and the associated R-loop dynamics, all are impacted by DNA shape (read more..). We are using the spin-labeling methods, in conjunction with other biophysical tools, are being applied to investigate DNA shape features of long, free duplexes under different topological constraints, and to elucidate impacts of DNA shape on Cas9 and Cas12a target interrogation.