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1. An Extended DNA Binding Domain of the Estrogen Receptor Alpha Directly Interacts with RNAs in Vitro

   https://doi.org/10.1021/acs.biochem.2c00536  

   Steiner, Halley R.; Lammer, Nickolaus C.; Batey, Robert T.; Wuttke, Deborah S. (October 2022, Biochemistry)

   Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs in vivo. Whether these interactions are direct or indirect remains unknown, which limits the ability to understand the extent, specificity, and biological role of ERα-RNA binding. Here we demonstrate that an extended DNA-binding domain of ERα directly binds a wide range of RNAs in vitro with structural specificity. ERα binds RNAs that adopt a range of hairpin-derived structures independent of sequence, while interacting poorly with single- and double-stranded RNA. RNA affinities are only 4-fold weaker than consensus dsDNA and significantly tighter than nonconsensus dsDNA sequences. Moreover, RNA binding is competitive with DNA binding. Together, these data show that ERα utilizes an extended DNA-binding domain to achieve a high-affinity/low-specificity mode for interacting with RNA. 

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2. MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

   https://doi.org/10.1038/s41586-023-06889-6  

   Cho, Min-Guk; Kumar, Rashmi J; Lin, Chien-Chu; Boyer, Joshua A; Shahir, Jamshaid A; Fagan-Solis, Katerina; Simpson, Dennis A; Fan, Cheng; Foster, Christine E; Goddard, Anna M; et al (January 2024, Nature)

   Abstract Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS–STING-mediated signalling and tumour suppression^1–3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)^4–10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11–RAD50–NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1–RIPK3–MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation ofZBP1in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis. 

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3. A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double‐Stranded λ DNA Reporter

   https://doi.org/10.1002/anie.202213920  

   Mohammad, Noor; Katkam, Shrinivas S.; Wei, Qingshan (November 2022, Angewandte Chemie International Edition)

   Abstract CRISPR‐based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR‐Cas12a‐based sensing platform to detect ssDNA targets by sizing double‐stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massivetrans‐nuclease activity of Cas12a toward λ DNA is due to the presence of single‐stranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes thetrans‐cleavage activity and helps achieve sub‐picomolar detection sensitivity, ≈100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters. 

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4. Triplex‐Forming Peptide Nucleic Acids with Extended Backbones

   https://doi.org/10.1002/cbic.202000432  

   Kumar, Vipin; Brodyagin, Nikita; Rozners, Eriks (August 2020, ChemBioChem)

   Abstract Peptide nucleic acid (PNA) forms a triple helix with double‐stranded RNA (dsRNA) stabilized by a hydrogen‐bonding zipper formed by PNA's backbone amides (N−H) interacting with RNA phosphate oxygens. This hydrogen‐bonding pattern is enabled by the matching ∼5.7 Å spacing (typical for A‐form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one −CH₂− group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B‐form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA‐DNA triplexes compared to PNA‐RNA. To test this hypothesis, we synthesized triplex‐forming PNAs that had the pseudopeptide backbones extended by an additional −CH₂− group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A‐form‐like conformations when binding double‐stranded nucleic acids. It appears that the original six‐atom‐long PNA backbone is an almost perfect fit for binding to A‐form nucleic acids. 

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5. Filament assembly underpins the double-stranded DNA specificity of AIM2-like receptors

   https://doi.org/10.1093/nar/gkad090  

   Garg, Archit; Stallings, Christina M.; Sohn, Jungsan (March 2023, Nucleic Acids Research)

   Abstract Upon sensing cytosolic- and/or viral double-stranded (ds)DNA, absent-in-melanoma-2 (AIM2)-like-receptors (ALRs) assemble into filamentous signaling platforms to initiate inflammatory responses. The versatile yet critical roles of ALRs in host innate defense are increasingly appreciated; however, the mechanisms by which AIM2 and its related IFI16 specifically recognize dsDNA over other nucleic acids remain poorly understood (i.e. single-stranded (ss)DNA, dsRNA, ssRNA and DNA:RNA hybrid). Here, we find that although AIM2 can interact with various nucleic acids, it preferentially binds to and assembles filaments faster on dsDNA in a duplex length-dependent manner. Moreover, AIM2 oligomers assembled on nucleic acids other than dsDNA not only display less ordered filamentous structures, but also fail to induce the polymerization of downstream ASC. Likewise, although showing broader nucleic acid selectivity than AIM2, IFI16 binds to and oligomerizes most readily on dsDNA in a duplex length-dependent manner. Nevertheless, IFI16 fails to form filaments on single-stranded nucleic acids and does not accelerate the polymerization of ASC regardless of bound nucleic acids. Together, we reveal that filament assembly is integral to nucleic acid distinction by ALRs. 

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