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1. 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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2. 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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3. Nonenzymatic RNA copying with a potentially primordial genetic alphabet

   https://doi.org/10.1073/pnas.2505720122  

   Fang, Ziyuan; Jia, Xiwen; Xing, Yanfeng; Szostak, Jack W (May 2025, Proceedings of the National Academy of Sciences)

   Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U because of the stronger G:C vs. A:U base pair and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s²U) and inosine (I) form by deamination of 2-thiocytidine (s²C) and A, respectively. We used thermodynamic and crystallographic analyses to compare the I:s²C and A:s²U base pairs. We find that the I:s²C base pair is isomorphic and isoenergetic with the A:s²U base pair. The I:s²C base pair is weaker than a canonical G:C base pair, while the A:s²U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s²U, s²C, I, and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s²C and s²U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s²U, s²C, I, and A and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying. 

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4. Design and Characterization of Compact, Programmable, Multistranded Nonimmunostimulatory Nucleic Acid Nanoparticles Suitable for Biomedical Applications

   https://doi.org/10.1021/acs.biochem.3c00615  

   Brumett, Ross; Danai, Leyla; Coffman, Abigail; Radwan, Yasmine; Teter, Megan; Hayth, Hannah; Doe, Erwin; Pranger, Katelynn; Thornburgh, Sable; Dittmer, Allison; et al (February 2024, Biochemistry)

   We report a thorough investigation of the role of single-stranded thymidine (ssT) linkers in the stability and flexibility of minimal, multistranded DNA nanostructures. We systematically explore the impact of varying the number of ssTs in three-way junction motifs (3WJs) on their formation and properties. Through various UV melting experiments and molecular dynamics simulations, we demonstrate that while the number of ssTs minimally affects thermodynamic stability, the increasing ssT regions significantly enhance the structural flexibility of 3WJs. Utilizing this knowledge, we design triangular DNA nanoparticles with varying ssTs, all showing exceptional assembly efficiency except for the 0T triangle. All triangles demonstrate enhanced stability in blood serum and are nonimmunostimulatory and nontoxic in mammalian cell lines. The 4T 3WJ is chosen as the building block for constructing other polygons due to its enhanced flexibility and favorable physicochemical characteristics, making it a versatile choice for creating cost-effective, stable, and functional DNA nanostructures that can be stored in the dehydrated forms while retaining their structures. Our study provides valuable insights into the design and application of nucleic acid nanostructures, emphasizing the importance of understanding stability and flexibility in the realm of nucleic acid nanotechnology. Our findings suggest the intricate connection between these ssTs and the structural adaptability of DNA 3WJs, paving the way for more precise design and engineering of nucleic acid nanosystems suitable for broad biomedical applications. 

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5. RNA-based strategy of enteroviral genome replication

   https://doi.org/10.1063/4.0000546  

   Das, Naba Krishna; Vogt, Jeff; Patel, Alisha; Banna, Hasan Al; Koirala, Deepak (March 2025, Structural Dynamics)

   Enteroviruses comprise a significant class of pathogens that cause various human diseases ranging from the common cold to poliomyelitis, acute flaccid paralysis, and myocarditis. The enteroviral (+)-strand RNA genome replication, essential for viral proliferation, has been proposed to depend on RNA structures at the genome’s extreme 5ʹ end. Such replication-linked RNAs (REPLRs) recruit essential proteins, the host poly-C binding protein 2 (PCBP2) and viral 3CD protein (precursor of the viral protease 3C and RNA-dependent RNA polymerase D) during genome replication, but the tertiary structures and mechanisms of the enteroviral REPLRs are mainly unknown. Recently, we have determined the crystal structures of CVB3, RVB14, and RVC15 REPLRs, revealing their highly conserved H-type four-way junction folds with co-axially stacked subdomains. The sA helix stacks on the sD helix, the sB helix on the sC helix, and the structure forms a unique long-range A•C•U base-triple between the sC- loop and the sD-helix. These conserved features enabled us to perform the structural prediction of additional enteroviral REPLRs through a homology modeling approach. The structure-guided binding studies with viral 3C revealed its primary binding site being the sD tetra-loop and a dinucleotide bulge. Moreover, the human PCBP2 binding studies revealed two binding sites for this protein – the sB loop and 3ʹ spacer, which collectively bind a single PCBP2 cooperatively. We also showed that the A•C•U base-triple disruption did not affect the 3C binding but did abrogate the PCBP2 interactions with the REPLR, suggesting a crucial role of this tertiary interaction in positioning the 3C and PCBP2 binding sites within the enteroviral REPLRs. Furthermore, oligonucleotides complementary to the spacer region diminished REPLR-PCBP2 interactions, highlighting the crucial function of this single-stranded segment in recruiting PCBP2. This insight sheds light on the potential for developing therapeutics to combat enteroviral infections by targeting this replication platform. 

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