Using the David H. Murdock Research Institute (DHMRI) 950 MHz NMR, Irina Nesmelova, PhD, assistant professor in the UNC Charlotte department of Physics and Optical Science, studied the atomic-level structure of the Sleeping Beauty (SB) transposase and its interactions with DNA. SB is the most used DNA transposon in genetic applications that currently is in clinical trials for human gene therapy.
Through the use of the DHMRI’s 950 MHz NMR, she obtained the first experimental structural information on SB transposase. She found that the “N-terminal PAI sub-domain forms a compact, three-helical structure with a helix-turn-helix motif, which is one of common DNA-binding motifs in proteins.” This structure verified earlier predictions of helical conformation and identified the exact location of helices and the helix-turn-helix motif. Her research confirmed that the “SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA,” and that the “PAI subdomain . . . has a dominant role in transposase’s attachment to the inverted terminal repeats of the transposon DNA.”
Her study also revealed that the DNA-binding domain of SB transposase does not form a stable structure at physiologic conditions, and that only folded PAI subdomain binds the transposon DNA. These findings open ways to rationally increase the efficiency of SB transposon through increasing the structural stability of the DNA-binding domain of SB transposase.
The research showed structural characteristics of SB transposase and contributes to the goal of improving its effectiveness as a tool for gene therapy. These results were published in the paper “NMR structural analysis of Sleeping Beauty Transposase binding to DNA” in the journal Protein Science, and in the paper “The Folding of the Specific DNA Recognition Subdomain of the Sleeping Beauty Transposase Is Temperature-Dependent and Is Required for Its Binding to the Transposon DNA” published in PlOS ONE.