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1. Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single-Stranded RNA

   https://doi.org/10.1021/acs.est.1c01255  

   Zhang, Ke ; Hodge, Joseph ; Chatterjee, Anamika ; Moon, Tae Seok ; Parker, Kimberly M. ( May 2021 , Environmental science technology)

   null (Ed.)

   Phosphodiester bonds in the backbones of double-stranded (ds)RNA and single-stranded (ss)RNA are known to undergo alkaline hydrolysis. Consequently, dsRNA agents used in emerging RNA interference (RNAi) products have been assumed to exhibit low chemical persistence in solutions. However, the impact of the duplex structure of dsRNA on alkaline hydrolysis has not yet been evaluated. In this study, we demonstrated that dsRNA undergoes orders-of-magnitude slower alkaline hydrolysis than ssRNA. Furthermore, we observed that dsRNA remains intact for multiple months at neutral pH, challenging the assumption that dsRNA is chemically unstable. In systems enabling both enzymatic degradation and alkaline hydrolysis of dsRNA, we found that increasing pH effectively attenuated enzymatic degradation without inducing alkaline hydrolysis that was observed for ssRNA. Overall, our findings demonstrated, for the first time, that key degradation pathways of dsRNA significantly differ from those of ssRNA. Consideration of the unique properties of dsRNA will enable greater control of dsRNA stability during the application of emerging RNAi technology and more accurate assessment of its fate in environmental and biological systems, as well as provide insights into broader application areas including dsRNA isolation, detection and inactivation of dsRNA viruses, and prebiotic molecular evolution. 

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2. Charge transport in individual short base stacked single-stranded RNA molecules

   https://doi.org/10.1038/s41598-023-46263-0  

   Chandra, Subrata ; Williams, Ajoke ; Maksudov, Farkhad ; Kliuchnikov, Evgenii ; Pattiya Arachchillage, Keshani G. G. ; Piscitelli, Patrick ; Castillo, Aderlyn ; Marx, Kenneth A. ; Barsegov, Valeri ; Artes Vivancos, Juan M. ( November 2023 , Scientific Reports)

   Charge transport in biomolecules is crucial for many biological and technological applications, including biomolecular electronics devices and biosensors. RNA has become the focus of research because of its importance in biomedicine, but its charge transport properties are not well understood. Here, we use the Scanning Tunneling Microscopy-assisted molecular break junction method to measure the electrical conductance of particular 5-base and 10-base single-stranded (ss) RNA sequences capable of base stacking. These ssRNA sequences show single-molecule conductance values around$$10^{-3}G_0$$${10}^{-3}{G}_0$($$G_0= 2e^2/h$$$G_0=2e^2/h$), while equivalent-length ssDNAs result in featureless conductance histograms. Circular dichroism (CD) spectra and MD simulations reveal the existence of extended ssRNA conformations versus folded ssDNA conformations, consistent with their different electrical behaviors. Computational molecular modeling and Machine Learning-assisted interpretation of CD data helped us to disentangle the structural and electronic factors underlying CT, thus explaining the observed electrical behavior differences. RNA with a measurable conductance corresponds to sequences with overall extended base-stacking stabilized conformations characterized by lower HOMO energy levels delocalized over a base-stacking mediating CT pathway. In contrast, DNA and a control RNA sequence without significant base-stacking tend to form closed structures and thus are incapable of efficient CT.

    

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3. Structural basis for Cas9 off-target activity.

   M. Pacesa, C-H. Lin ( October 2022 , Cell)

   John Pham, Ph.D. Editor-in-Chief (Ed.)

   The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non-canonical base-pairing interactions and preservation of base stacking within the guide–off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple non-canonical base pairs rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms. 

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4. Target-specific requirements for RNA interference can be explained by a single regulatory network

   Knudsen, D. ; Raman, P. ; Ettefa, F. ; De Ravin, L. ; Jose, A. ( February 2023 , bioRxiv)

   Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode C. elegans can selectively impair the silencing of some genes, raising the possibility of gene-specific specialization of the RNAi mechanism. Here we dissect the silencing of two somatic genes in detail to show that such selective regulation can be explained by a single network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one gene, but each is singly required for silencing the other gene. While numerous features could distinguish one gene from another, quantitative models suggest that, for the same steady state abundance of mRNA, genes with higher rates of mRNA production are more difficult to knockdown with a single dose of dsRNA and recovery from knockdown can occur if all intermediates of RNA silencing undergo turnover. Consistent with such dissipation of RNA silencing, animals recover after silencing by a pulse of dsRNA and show restricted production of templates for amplifying small RNAs. The loss of NRDE-3 can be overcome by enhancing dsRNA processing, which supports a quantitative contribution of this regulator to RNA silencing. These insights explain selectivity in the requirements for specific regulators without invoking different mechanisms for different sets of genes. 

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5. Development of Cu 2+ -Based Distance Methods and Force Field Parameters for the Determination of PNA Conformations and Dynamics by EPR and MD Simulations

   https://doi.org/10.1021/acs.jpcb.0c05509  

   Gamble Jarvi, Austin ; Sargun, Artur ; Bogetti, Xiaowei ; Wang, Junmei ; Achim, Catalina ; Saxena, Sunil ( August 2020 , The Journal of Physical Chemistry B)

   Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu(II) complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV–vis and circular dichroism measurements and assess the efficacy of the Cu(II) label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu(II) label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu(II) EPR at X-band. These results present for the first time a rigid Cu(II) spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large. 

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