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1. DNA Nanostructures as Catalysts: Double Crossover Tile-Assisted 5′ to 5′ and 3′ to 3′ Chemical Ligation of Oligonucleotides

   Bardales A.C, Mills J.R. (January 2024, Bioconjugate chemistry)

   Accessibility of synthetic oligonucleotides and the success of DNA nanotechnology open a possibility to use DNA nanostructures for building sophisticated enzyme-like catalytic centers. Here we used a double DNA crossover (DX) tile nanostructure to enhance the rate, the yield, and the specificity of 5′−5′ ligation of two oligonucleotides with arbitrary sequences. The ligation product was isolated via a simple procedure. The same strategy was applied for the synthesis of 3′−3′ linked oligonucleotides, thus introducing a synthetic route to DNA and RNA with a switched orientation that is affordable by a low- resource laboratory. To emphasize the utility of the ligation products, we synthesized a circular structure formed from intramolecular complementarity that we named “an impossible DNA wheel” since it cannot be built from regular DNA strands by enzymatic reactions. Therefore, DX-tile nanostructures can open a route to producing useful chemical products that are unattainable via enzymatic synthesis. This is the first example of the use of DNA nanostructures as a catalyst. This study advocates for further exploration of DNA nanotechnology for building enzyme-like reactive systems. 

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2. Synthesis of 5‐Cyanomethyluridine (cnm ⁵ U) and 5‐Cyanouridine (cn ⁵ U) Phosphoramidites and Their Incorporation into RNA Oligonucleotides

   https://doi.org/10.1002/cpnc.114  

   Mao, Song; Tsai, Hsu‐Chun; Sheng, Jia (August 2020, Current Protocols in Nucleic Acid Chemistry)

   Abstract This article contains detailed synthetic protocols for preparation of 5‐cyanomethyluridine (cnm⁵U) and 5‐cyanouridine (cn⁵U) phosphoramidites. The synthesis of the cnm⁵U phosphoramidite building block starts with commercially available 5‐methyluridine (m⁵C), followed by bromination of the 5‐methyl group to install the cyano moiety using TMSCN/TBAF. The cn⁵U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5‐cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA‐based therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cnm⁵U and cn⁵U phosphoramidites Basic Protocol 2: Synthesis, purification, and characterization of cnm⁵U‐ and cn⁵U‐modified RNA oligonucleotides 

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3. A method for site-specifically tethering the enzyme urease to DNA origami with sustained activity

   https://doi.org/10.1371/journal.pone.0319790  

   Murphy, Ian; Bobilev, Keren; Hayakawa, Daichi; Ikonen, Eden; Videbæk, Thomas E; Dalal, Shibani; Ahmed, Wylie W; Ross, Jennifer L; Rogers, W Benjamin (April 2025, PLOS One)

   Signore, Giovanni (Ed.)

   Attaching enzymes to nanostructures has proven useful to the study of enzyme functionality under controlled conditions and has led to new technologies. Often, the utility and interest of enzyme-tethered nanostructures lie in how the enzymatic activity is affected by how the enzymes are arranged in space. Therefore, being able to conjugate enzymes to nanostructures while preserving the enzymatic activity is essential. In this paper, we present a method to conjugate single-stranded DNA to the enzyme urease while maintaining enzymatic activity. We show evidence of successful conjugation and quantify the variables that affect the conjugation yield. We also show that the enzymatic activity is unchanged after conjugation compared to the enzyme in its native state. Finally, we demonstrate the tethering of urease to nanostructures made using DNA origami with high site-specificity. Decorating nanostructures with enzymatically-active urease may prove to be useful in studying, or even utilizing, the functionality of urease in disciplines ranging from biotechnology to soft-matter physics. The techniques we present in this paper will enable researchers across these fields to modify enzymes without disrupting their functionality, thus allowing for more insightful studies into their behavior and utility. 

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4. Stabilizing DNA–Protein Co-Crystals via Intra-Crystal Chemical Ligation of the DNA

   https://doi.org/10.3390/cryst12010049  

   Ward, Abigail R.; Dmytriw, Sara; Vajapayajula, Ananya; Snow, Christopher D. (January 2022, Crystals)

   Protein and DNA co-crystals are most commonly prepared to reveal structural and functional details of DNA-binding proteins when subjected to X-ray diffraction. However, biomolecular crystals are notoriously unstable in solution conditions other than their native growth solution. To achieve greater application utility beyond structural biology, biomolecular crystals should be made robust against harsh conditions. To overcome this challenge, we optimized chemical DNA ligation within a co-crystal. Co-crystals from two distinct DNA-binding proteins underwent DNA ligation with the carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) under various optimization conditions: 5′ vs. 3′ terminal phosphate, EDC concentration, EDC incubation time, and repeated EDC dose. This crosslinking and DNA ligation route did not destroy crystal diffraction. In fact, the ligation of DNA across the DNA–DNA junctions was clearly revealed via X-ray diffraction structure determination. Furthermore, crystal macrostructure was fortified. Neither the loss of counterions in pure water, nor incubation in blood serum, nor incubation at low pH (2.0 or 4.5) led to apparent crystal degradation. These findings motivate the use of crosslinked biomolecular co-crystals for purposes beyond structural biology, including biomedical applications. 

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5. Understanding the physical processes behind DNA-DNA proximity ligation assays

   https://doi.org/10.1101/2025.05.02.649190  

   Zubillaga_Herrera, Bernardo J; Das, Amit; Burack, Linden; Wang, Ailung; Di_Pierro, Michele (May 2025, bioRxiv)

   Abstract In the last decade, DNA-DNA proximity ligation assays opened powerful new ways to study the 3D organization of genomes and have become a mainstay experimental technology. Yet many aspects of these experiments remain poorly understood. We study the inner workings of DNA-DNA proximity ligation assays through numerical experiments and theoretical modeling. Chromosomes are modeled at nucleosome resolution and evolved in time via molecular dynamics. A virtual Hi-C experiment reproduces, in-silico, the different steps of the Hi-C protocol, including: crosslinking of chromatin to an underlying proteic matrix, enzymatic digestion of DNA, and subsequent proximity ligation of DNA open ends. The protocol is simulated on ensembles of different structures as well as individual structures, enabling the construction of ligation maps and the calculation of ligation probabilities as functions of genomic and Euclidean distance. The methods help to assess the effect of the many variables of the Hi-C experiment and of subsequent data processing methods on the quality of the final results. 

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