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1. Single-molecule detection of oligonucleotides using the fluorescent nucleobase analogue ABN

   https://doi.org/10.1039/d4sc07334g  

   Samaan, George N; Jimenez_Salinas, Andres; Bailie, Alexandra E; Grim, Julian; Cizmic, Julian M; Jones, Anita C; Lee, Youngkwang; Purse, Byron W (February 2025, Chemical Science)

   We investigate the fluorescent pyrimidine analogue ABN in duplex DNA oligonucleotides, showing that ABN is unique among fluorescent nucleobase analogues in enabling single-molecule fluorescence studies of oligonucleotides using standard equipment. 

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2. Non‐Chromatographic Purification of Synthetic RNA Using Bio‐Orthogonal Chemistry

   https://doi.org/10.1002/cpz1.247  

   He, Muhan; Wu, Xunshen; Mao, Song; Haruehanroengra, Phensinee; Khan, Irfan; Sheng, Jia; Royzen, Maksim (September 2021, Current Protocols)

   Abstract Solid‐phase synthesis of RNA oligonucleotides over 100 nt in length remains challenging due to the complexity of purification of the target strands from the failure sequences. This article describes a non‐chromatographic procedure that will enable routine solid‐phase synthesis and purification of long RNA strands. The optimized five‐step process is based on bio‐orthogonal inverse electron demand Diels‐Alder chemistry betweentrans‐cyclooctene (TCO) and tetrazine (Tz), and entails solid‐phase synthesis of RNA on a photo‐labile support. The target oligonucleotide strands are selectively tagged with Tz while on‐support. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach can be applied for purification of 76‐nt long tRNA and 101‐nt long sgRNA for CRISPR experiments. Purity of the isolated oligonucleotides should be evaluated using gel electrophoresis, while functional fidelity of the sgRNA should be confirmed using CRISPR‐Cas9 experiments. © 2021 Wiley Periodicals LLC. Basic Protocol: Five‐step non‐chromatographic purification of synthetic RNA oligonucleotides Support Protocol 1: Synthesis of the components that are required for the non‐chromatographic purification of long RNA oligonucleotides. Support Protocol 2: Solid‐phase RNA synthesis 

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3. Low‐scale syringe‐made synthesis of ¹⁵ N‐labeled oligonucleotides

   https://doi.org/10.1002/jlcr.4000  

   Brož, Břetislav; Tureček, František; Marek, Aleš (September 2022, Journal of Labelled Compounds and Radiopharmaceuticals)

   Fast and reasonable low‐scale (200 nmol) syringe‐made synthesis of¹⁵N‐labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid‐phase controlled pore glass synthesis column technique via the phosphoramidite method. Twenty‐four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry. 

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4. Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides

   https://doi.org/10.3390/molecules25204659  

   Ochoa, Steven; Milam, Valeria T. (October 2020, Molecules)

   null (Ed.)

   In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides. 

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5. Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides

   https://doi.org/10.1093/nar/gkad439  

   Ding, Dian; Zhang, Stephanie J.; Szostak, Jack W. (May 2023, Nucleic Acids Research)

   Abstract The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5′–5′ 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5′–5′ 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life. 

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