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1. Improved Triplex‐Forming Isoorotamide PNA Nucleobases for A−U Recognition of RNA Duplexes**

   https://doi.org/10.1002/chem.202302390  

   Talbott, John M. ; Tessier, Brandon R. ; Harding, Emily E. ; Walby, Grant D. ; Hess, Kyle J. ; Baskevics, Vladislavs ; Katkevics, Martins ; Rozners, Eriks ; MacKay, James A. ( October 2023 , Chemistry – A European Journal)

   Four new isoorotamide (Io)‐containing PNA nucleobases have been designed for A−U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A−U in a PNA:RNA₂triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A‐binding nucleobases. Molecular dynamics simulations provided further insights into binding ofIobases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomerIo8additionally gave the highest affinity observed for an A‐binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.

    

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2. Nucleobase and Linker Modification for Triple‐Helical Recognition of Pyrimidines in RNA Using Peptide Nucleic Acids

   https://doi.org/10.1002/cbic.202300291  

   Kumpina, Ilze ; Baskevics, Vladislavs ; Nguyen, Khoi D. ; Katkevics, Martins ; Rozners, Eriks ( July 2023 , ChemBioChem)

   Triple‐helical recognition of any sequence of double‐stranded RNA requires high affinity Hoogsteen hydrogen binding to pyrimidine interruptions of polypurine tracts. Because pyrimidines have only one hydrogen bond donor/acceptor on Hoogsteen face, their triple‐helical recognition is a formidable problem. The present study explored various five‐membered heterocycles and linkers that connect nucleobases to backbone of peptide nucleic acid (PNA) to optimize formation of X•C‐G and Y•U‐A triplets. Molecular modeling and biophysical (UV melting and isothermal titration calorimetry) results revealed a complex interplay between the heterocyclic nucleobase and linker to PNA backbone. While the five‐membered heterocycles did not improve pyrimidine recognition, increasing the linker length by four atoms provided promising gains in binding affinity and selectivity. The results suggest that further optimization of heterocyclic bases with extended linkers to PNA backbone may be a promising approach to triple‐helical recognition of RNA.

    

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3. Triplex‐Forming Peptide Nucleic Acids with Extended Backbones

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

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

   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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4. N‐Terminal Protein Labeling with N ‐Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein‐Protein Interactions Using Labeled Protein

   https://doi.org/10.1002/cpz1.14  

   Jiang, Hanjie ; Cole, Philip A. ( January 2021 , Current Protocols)

   Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein‐protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy‐to‐apply protocol for high‐stoichiometric, site‐specific labeling of a protein at its N‐terminus withN‐hydroxysuccinimide (NHS) esters as a means to measure protein‐protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent‐labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2‐E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC.

   This article was corrected on 18 July 2022. See the end of the full text for details.

   Basic Protocol 1: Labeling a protein of interest at its N‐terminus with NHS esters through stepwise reaction

   Alternate Protocol: Labeling a protein of interest at its N‐terminus with NHS esters through a one‐pot reaction

   Basic Protocol 2: Purifying the N‐terminal fluorescent‐labeled protein and determining its concentration and labeling efficiency

   Basic Protocol 3: Using MST to determine the binding affinity of an N‐terminal fluorescent‐labeled protein to a binding partner.

   Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay

    

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5. Cage Match: Comparing the Anion Binding Ability of Isostructural Versus Isofunctional Pairs of Metal‐Organic Nanocages

   https://doi.org/10.1002/chem.202303013  

   Dutton, Kaitlyn G. ; Jones, Taro J. ; Emge, Thomas J. ; Lipke, Mark C. ( November 2023 , Chemistry – A European Journal)

   Affinities of six anions (mesylate, acetate, trifluoroacetate,p‐toluenecarboxylate,p‐toluenesulfonate, and perfluorooctanoate) for three related Pt²⁺‐linked porphyrin nanocages were measured to probe the influence of different noncovalent recognition motifs (e. g., hydrogen bonding, electrostatics, π bonding) on anion binding. Two new hosts of M₆L₃¹²⁺(1b) and M₄L₂⁸⁺(2) composition (M=(en)Pt²⁺, L=(3‐py)₄porphyrin) were prepared in a one‐pot synthesis and allowed comparison of hosts that differ in structure while maintaining similar N−H hydrogen‐bond donor ability. Comparisons of isostructural hosts that differ in hydrogen‐bonding ability were made between1band a related M₆L₃¹²⁺nanoprism (1a, M=(tmeda)Pt²⁺) that lacks N−H groups. Considerable variation in association constants (K₁=1.6×10³ M⁻¹to 1.3×10⁸ M⁻¹) and binding mode (exovs.endo) were found for different host–guest combinations. Strongest binding was seen betweenp‐toluenecarboxylate and1b, but surprisingly, association of this guest with1awas only slightly weaker despite the absence of NH⋅⋅⋅O interactions. The high affinity betweenp‐toluenecarboxylate and1acould be turned off by protonation, and this behavior was used to toggle between the binding of this guest and the environmental pollutant perfluorooctanoate, which otherwise has a lower affinity for the host.

    

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