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1. Understanding DNA interactions in crowded environments with a coarse-grained model

   https://doi.org/10.1093/nar/gkaa854  

   Hong, Fan; Schreck, John S; Šulc, Petr (October 2020, Nucleic Acids Research)

   null (Ed.)

   Abstract Nucleic acid interactions under crowded environments are of great importance for biological processes and nanotechnology. However, the kinetics and thermodynamics of nucleic acid interactions in a crowded environment remain poorly understood. We use a coarse-grained model of DNA to study the kinetics and thermodynamics of DNA duplex and hairpin formation in crowded environments. We find that crowders can increase the melting temperature of both an 8-mer DNA duplex and a hairpin with a stem of 6-nt depending on the excluded volume fraction of crowders in solution and the crowder size. The crowding induced stability originates from the entropic effect caused by the crowding particles in the system. Additionally, we study the hybridization kinetics of DNA duplex formation and the formation of hairpin stems, finding that the reaction rate kon is increased by the crowding effect, while koff is changed only moderately. The increase in kon mostly comes from increasing the probability of reaching a transition state with one base pair formed. A DNA strand displacement reaction in a crowded environment is also studied with the model and we find that rate of toehold association is increased, with possible applications to speeding up strand displacement cascades in nucleic acid nanotechnology. 

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2. Toehold clipping: A mechanism for remote control of DNA strand displacement

   https://doi.org/10.1093/nar/gkac1152  

   Faheem, Hiba; Mathivanan, Johnsi; Talbot, Hannah; Zeghal, Hana; Vangaveti, Sweta; Sheng, Jia; Chen, Alan A.; Chandrasekaran, Arun Richard (December 2022, Nucleic Acids Research)

   Abstract The ability to create stimuli-responsive DNA nanostructures has played a prominent role in dynamic DNA nanotechnology. Primary among these is the process of toehold-based strand displacement, where a nucleic acid molecule can act as a trigger to cause conformational changes in custom-designed DNA nanostructures. Here, we add another layer of control to strand displacement reactions through a 'toehold clipping' process. By designing DNA complexes with a photocleavable linker-containing toehold or an RNA toehold, we show that we can use light (UV) or enzyme (ribonuclease) to eliminate the toehold, thus preventing strand displacement reactions. We use molecular dynamics simulations to analyze the structural effects of incorporating a photocleavable linker in DNA complexes. Beyond simple DNA duplexes, we also demonstrate the toehold clipping process in a model DNA nanostructure, by designing a toehold containing double-bundle DNA tetrahedron that disassembles when an invading strand is added, but stays intact after the toehold clipping process even in the presence of the invading strand. This work is an example of combining multiple physical or molecular stimuli to provide additional remote control over DNA nanostructure reconfiguration, advances that hold potential use in biosensing, drug delivery or molecular computation. 

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3. Precise in-field molecular diagnostics of crop diseases by smartphone-based mutation-resolved pathogenic RNA analysis

   https://doi.org/10.1038/s41467-023-39952-x  

   Zhang, Ting; Zeng, Qingdong; Ji, Fan; Wu, Honghong; Ledesma-Amaro, Rodrigo; Wei, Qingshan; Yang, Hao; Xia, Xuhan; Ren, Yao; Mu, Keqing; et al (December 2023, Nature Communications)

   NA (Ed.)

   Abstract Molecular diagnostics for crop diseases can guide the precise application of pesticides, thereby reducing pesticide usage while improving crop yield, but tools are lacking. Here, we report an in-field molecular diagnostic tool that uses a cheap colorimetric paper and a smartphone, allowing multiplexed, low-cost, rapid detection of crop pathogens. Rapid nucleic acid amplification-free detection of pathogenic RNA is achieved by combining toehold-mediated strand displacement with a metal ion-mediated urease catalysis reaction. We demonstrate multiplexed detection of six wheat pathogenic fungi and an early detection of wheat stripe rust. When coupled with a microneedle for rapid nucleic acid extraction and a smartphone app for results analysis, the sample-to-result test can be completed in ~10 min in the field. Importantly, by detecting fungal RNA and mutations, the approach allows to distinguish viable and dead pathogens and to sensitively identify mutation-carrying fungicide-resistant isolates, providing fundamental information for precision crop disease management. 

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4. Significant Differences in RNA Structure Destabilization by HIV-1 Gag∆p6 and NCp7 Proteins

   https://doi.org/10.3390/v12050484  

   McCauley, Micah J.; Rouzina, Ioulia; Li, Jasmine; Núñez, Megan E.; Williams, Mark C. (May 2020, Viruses)

   null (Ed.)

   Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and Gag∆p6 destabilize the TAR hairpin, Gag∆p6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike Gag∆p6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~104. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion. 

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5. Synthetic Genes For Dynamic Regulation Of DNA‐Based Receptors

   https://doi.org/10.1002/ange.202319382  

   Sorrentino, Daniela; Ranallo, Simona; Nakamura, Eiji; Franco, Elisa; Ricci, Francesco (April 2024, Angewandte Chemie)

   Abstract We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA‐based receptors: a triplex‐forming DNA complex, an ATP‐binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling. 

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