DNA dynamics in biology and technology

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DNA was long viewed as a passive carrier of genetic information whose biologically relevant form was the canonical, B-form helical duplex. However, it is becoming increasingly clear that DNA dynamically adopts a wide variety of non-canonical structures that are implicated in fundamental processes such as chromosome stability, cell senescence, and gene expression, and have shown promise in therapeutics and technology. Some of the most-studied non-canonical DNA forms are guanine quadruplexes (G4s) and i-motifs (iMs), four-stranded structures formed by dG- and dC-rich DNA, respectively. G4s and iMs are often conformationally heterogeneous, leading to highly complex dynamical behaviour. In most cases, we are not able to predict the structures or dynamics experienced by naturally occurring G- or C-rich DNA with any precision, nor can we anticipate the effects of oxidative damage and epigenetic modifications or interactions with enzymes, such as helicases and polymerases. Furthermore, many of the current functional assays are largely qualitative. Thus, even though over 350,000 putative G4 forming sequences have been identified in the human genome, their biological functions are overwhelmingly unknown. To address this gap in our understanding, we need new quantitative assays to better characterize DNA structure, dynamics, and function, and to elucidate general sequence-structure-dynamics-function relationships. This information is critical for understanding the role of non-canonical DNA in health and disease, and for fully exploiting DNA in biotechnological applications. To address this challenge, our short-term objectives are to develop a suite of new biophysical tools to quantitatively characterize DNA structure, dynamics and function with a higher level of detail than is currently possible. These include (i) a highly sensitive assay for measuring the blockage of polymerases by G4s and iMs; (ii) a method to precisely determine the numbers folded and unfolded residues in a DNA chain; and (iii) an approach for gathering structural information at the single-molecule level for highly heterogeneous structural ensembles. We will use these new tools to elucidate quantitative sequence-structure-dynamics-function relationships for some important classes of non-canonical DNA: telomeric and promoter G4s and iMs, aptamers, as well as dG- and dC- rich regions that we predict to be hugely conformationally heterogeneous with upwards of thousands or tens of thousands of different structural isomers. The long-term vision of our research involves (i) understanding and accurately predicting the structures, dynamics, and functional interactions of naturally occurring non-canonical DNA based on the primary nucleotide sequences, enabling the rational design of therapeutics targeting DNA, and (ii) generating improved DNA-based biosensors, therapeutics, and drug delivery vehicles with novel biophysical properties based on binding cooperativity.