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Nucleobase mimicking small molecules able to reconfigure DNA are a recently discovered strategy that promises to extend the structural and functional diversity of nucleic acids. However, only simple, unfunctionalized molecules such as cyanuric acid and melamine have so far been used in this approach. In this work, we show that the addition of substituted cyanuric acid molecules can successfully program polyadenine strands to assemble into supramolecular fibers. Unlike conventional DNA nanostructure functionalization, which typically end-labels DNA strands, our approach incorporates functional groups into DNA with high density using small molecules and results in new DNA triple helices coated with alkylamine or alcohol units that grow into micrometer-long fibers. We find that small changes in the small molecule functional group can result in large structural and energetic variation in the overall assembly. A combination of circular dichroism, atomic force microscopy, molecular dynamics simulations, and a new thermodynamic method, transient equilibrium mapping, elucidated the molecular factors behind these large changes. In particular, we identify substantial DNA sugar and phosphate group deformations to accommodate a hydrogen bond between the phosphate and the small-molecule functional groups, as well as a critical chain length of the functional group which switches this interaction from intra- to interfiber. These parameters allow the controlled formation of hierarchical, hybrid DNA assemblies simply through the addition and variation of small, functionalized molecules.

The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown. To address this question, we studied two different G-quadruplexes, selecting a single conformation by blocking hydrogen bonding with photolabile protection groups. Upon irradiation, the block can be released and the kinetics of re-equilibration to the native conformational equilibrium can be determined by time-resolved NMR. We compared the NMR-derived refolding kinetics with data derived from thermal hysteresis folding kinetic experiments and found excellent agreement. The outlined methodological approach allows separation of K + -induced G-quadruplex formation and subsequent refolding and provides key insight into rate-limiting steps of G-quadruplex conformational dynamics.

G-quadruplexes (GQs) are four-stranded nucleic acid secondary structures formed by guanosine (G)-rich DNA and RNA sequences. It is becoming increasingly clear that cellular processes including gene expression and mRNA translation are regulated by GQs. GQ structures have been extensively characterized, however little attention to date has been paid to their conformational dynamics, despite the fact that many biological GQ sequences populate multiple structures of similar free energies, leading to an ensemble of exchanging conformations. The impact of these dynamics on biological function is currently not well understood. Recently, structural dynamics have been demonstrated to entropically stabilize GQ ensembles, potentially modulating gene expression. Transient, low-populated states in GQ ensembles may additionally regulate nucleic acid interactions and function. This review will underscore the interplay of GQ dynamics and biological function, focusing on several dynamic processes for biological GQs and the characterization of GQ dynamics by nuclear magnetic resonance (NMR) spectroscopy in conjunction with other biophysical techniques. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman., (Copyright © 2017 Elsevier B.V. All rights reserved.)

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood. For example, when there are surplus guanosines in some of the tracts, they can slide with respect to one another, a process we term G-register (GR) exchange. These motions could in principle entropically stabilize the folded state, crucially benefitting GQs as their stabilities are closely tied to biological function. We have developed a method for characterizing GR exchange where each isomer in the wild-type conformational ensemble is trapped by mutation and thermal denaturation data for the set of trapped mutants and wild-type are analyzed simultaneously. This yields GR isomer populations as a function of temperature, quantifies conformational entropy and sheds light on correlated sliding motions of the G-tracts. We measured entropic stabilizations from GR exchange up to 14.3 ± 1.6 J mol(-1) K(-1), with melting temperature increases up to 7.3 ± 1.6°C. Furthermore, bioinformatic analysis suggests a majority of putative human GQ sequences are capable of GR exchange, pointing to the generality of this phenomenon., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Self‐replication of nucleic acids in the absence of enzymes represents an important and poorly understood step in the origin of life as such reported systems are strongly hindered by product inhibition. Studying one of the few successful examples of enzymatic DNA self‐replication based on a simple ligation chain reaction, lesion‐induced DNA amplification (LIDA), can shed light on how this fundamental process may have originally evolved. To identify the unknown factors that lead LIDA to overcome product inhibition we have employed isothermal titration calorimetry and global fitting of time‐dependent ligation data to characterize the individual steps of the amplification process. We find that incorporating the abasic lesion into one of the four primers substantially decreases the stability difference between the product and intermediate complexes compared with complexes without the abasic group. In the presence of T4 DNA ligase this stability gap is further reduced by two orders of magnitude revealing that the ligase also helps overcome product inhibition. Kinetic simulations reveal that the intermediate complex stability and the magnitude of the ligation rate constant significantly impact the rate of self‐replication, suggesting that catalysts that both facilitate ligation and stabilize the intermediate complex might be a route to efficient nonenzymatic replication. [ABSTRACT FROM AUTHOR]