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1. Crystal structure of an RNA/DNA strand exchange junction

   https://doi.org/10.1371/journal.pone.0263547  

   Cofsky, Joshua C. ; Knott, Gavin J. ; Gee, Christine L. ; Doudna, Jennifer A. ( April 2022 , PLOS ONE)

   Kursula, Petri (Ed.)

   Short segments of RNA displace one strand of a DNA duplex during diverse processes including transcription and CRISPR-mediated immunity and genome editing. These strand exchange events involve the intersection of two geometrically distinct helix types—an RNA:DNA hybrid (A-form) and a DNA:DNA homoduplex (B-form). Although previous evidence suggests that these two helices can stack on each other, it is unknown what local geometric adjustments could enable A-on-B stacking. Here we report the X-ray crystal structure of an RNA-5′/DNA-3′ strand exchange junction at an anisotropic resolution of 1.6 to 2.2 Å. The structure reveals that the A-to-B helical transition involves a combination of helical axis misalignment, helical axis tilting and compression of the DNA strand within the RNA:DNA helix, where nucleotides exhibit a mixture of A- and B-form geometry. These structural principles explain previous observations of conformational stability in RNA/DNA exchange junctions, enabling a nucleic acid architecture that is repeatedly populated during biological strand exchange events. 

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2. Impact of bPNA Backbone Structural Constraints and Composition on Triplex Hybridization with DNA

   https://doi.org/10.1002/cbic.202100707  

   Munyaradzi, Oliver ; Rundell, Sarah ; Bong, Dennis ( February 2022 , ChemBioChem)

   We report herein a study on the impact of bifacial peptide nucleic acid (bPNA) amino acid composition and backbone modification on DNA binding. A series of bPNA backbone variants with identical net charge were synthesized to display either 4 or 6 melamine (M) bases. These bases form thymine‐melamine‐thymine (TMT) base‐triples, resulting in triplex hybrid stem structures with T‐rich DNAs. Analyses of 6 M bPNA‐DNA hybrids suggested that hybrid stability was linked to amino acid secondary structure propensities, prompting a more detailed study in shorter 4 M bPNAs. We synthesized 4 M bPNAs predisposed to adopt helical secondary structure via helix‐turn nucleation in 7‐residue bPNAs using double‐click covalent stapling. Generally, hybrid stability improved upon stapling, but amino acid composition had a more significant effect. We also pursued an alternative strategy for bPNA structural preorganization by incorporation of residues with strong backbone amide conformational preferences such as 4R‐ and 4S‐fluoroprolines. Notably, these derivatives exhibited an additional improvement in hybrid stability beyond both unsubstituted proline bPNA analogues and the helically patterned bPNAs. Overall, these findings demonstrate the tunability of bPNA‐DNA hybrid stability through bPNA backbone structural propensities and amino acid composition.

    

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3. Computational investigation of the impact of core sequence on immobile DNA four-way junction structure and dynamics

   https://doi.org/10.1093/nar/gkab1246  

   Adendorff, Matthew R. ; Tang, Guo Qing ; Millar, David P. ; Bathe, Mark ; Bricker, William P. ( December 2021 , Nucleic Acids Research)

   Immobile four-way junctions (4WJs) are core structural motifs employed in the design of programmed DNA assemblies. Understanding the impact of sequence on their equilibrium structure and flexibility is important to informing the design of complex DNA architectures. While core junction sequence is known to impact the preferences for the two possible isomeric states that junctions reside in, previous investigations have not quantified these preferences based on molecular-level interactions. Here, we use all-atom molecular dynamics simulations to investigate base-pair level structure and dynamics of four-way junctions, using the canonical Seeman J1 junction as a reference. Comparison of J1 with equivalent single-crossover topologies and isolated nicked duplexes reveal conformational impact of the double-crossover motif. We additionally contrast J1 with a second junction core sequence termed J24, with equal thermodynamic preference for each isomeric configuration. Analyses of the base-pair degrees of freedom for each system, free energy calculations, and reduced-coordinate sampling of the 4WJ isomers reveal the significant impact base sequence has on local structure, isomer bias, and global junction dynamics.

    

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4. Hybrid DNA/RNA nanostructures with 2′-5′ linkages

   https://doi.org/10.1039/d0nr05846g  

   Chandrasekaran, Arun Richard ; Mathivanan, Johnsi ; Ebrahimi, Parisa ; Vilcapoma, Javier ; Chen, Alan A. ; Halvorsen, Ken ; Sheng, Jia ( November 2020 , Nanoscale)

   null (Ed.)

   Nucleic acid nanostructures with different chemical compositions have shown utility in biological applications as they provide additional assembly parameters and enhanced stability. The naturally occurring 2′-5′ linkage in RNA is thought to be a prebiotic analogue and has potential use in antisense therapeutics. Here, we report the first instance of DNA/RNA motifs containing 2′-5′ linkages. We synthesized and incorporated RNA strands with 2′-5′ linkages into different DNA motifs with varying number of branch points (a duplex, four arm junction, double crossover motif and tensegrity triangle motif). Using experimental characterization and molecular dynamics simulations, we show that hybrid DNA/RNA nanostructures can accommodate interspersed 2′-5′ linkages with relatively minor effect on the formation of these structures. Further, the modified nanostructures showed improved resistance to ribonuclease cleavage, indicating their potential use in the construction of robust drug delivery vehicles with prolonged stability in physiological conditions. 

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5. Increasing valence pushes DNA nanostar networks to the isostatic point

   https://doi.org/10.1073/pnas.1819683116  

   Conrad, Nathaniel ; Kennedy, Tynan ; Fygenson, Deborah K. ; Saleh, Omar A. ( March 2019 , Proceedings of the National Academy of Sciences)

   The classic picture of soft material mechanics is that of rubber elasticity, in which material modulus is related to the entropic elasticity of flexible polymeric linkers. The rubber model, however, largely ignores the role of valence (i.e., the number of network chains emanating from a junction). Recent work predicts that valence, and particularly the Maxwell isostatic point, plays a key role in determining the mechanics of semiflexible polymer networks. Here, we report a series of experiments confirming the prominent role of valence in determining the mechanics of a model system. The system is based on DNA nanostars (DNAns): multiarmed, self-assembled nanostructures that form thermoreversible equilibrium gels through base pair-controlled cross-linking. We measure the linear and nonlinear elastic properties of these gels as a function of DNAns arm number, f, and concentration [DNAns]. We find that, as f increases from three to six, the gel’s high-frequency plateau modulus strongly increases, and its dependence on [DNAns] transitions from nonlinear to linear. Additionally, higher-valence gels exhibit less strain hardening, indicating that they have less configurational freedom. Minimal strain hardening and linear dependence of shear modulus on concentration at high f are consistent with predictions for isostatic systems. Evident strain hardening and nonlinear concentration dependence of shear modulus suggest that the low-f networks are subisostatic and have a transient, potentially fractal percolated structure. Overall, our observations indicate that network elasticity is sensitive both to entropic elasticity of network chains and to junction valence, with an apparent isostatic point$5<f_c\le 6$in agreement with the Maxwell prediction.

    

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