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1. 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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2. Comparison of transcriptional activity profiling by metabolic labeling or nuclear RNA sequencing

   https://doi.org/10.1111/tpj.70401  

   Wear, E_E; Mickelson‐Young, L.; Bass, H_W; Hanley‐Bowdoin, L.; Thompson, W_F; Concia, L. (August 2025, The Plant Journal)

   SUMMARY The application of high‐throughput sequencing to cellular transcriptome profiling (RNA‐seq) has enabled significant advances in our understanding of gene expression in plants. However, conventional RNA‐seq data reports mainly cytoplasmic transcript abundance rather than actual transcription rates. As a result, it is less sensitive to detect unstable and low‐abundance nuclear RNA species, such as long non‐coding RNAs, and is less directly connected to chromatin features and processes such as DNA replication. To bridge this gap, several protocols have been established to profile newly synthesized RNA in plants and other eukaryotes. These protocols can be technically challenging and present their own difficulties and limitations. Here we analyze newly synthesized nuclear RNA metabolically labeledin vivowith 5‐ethynyl uridine (EU‐nuclear RNA) in maize (Zea maysL.) root tips and compare it with the entire nuclear RNA population. We also compare both nuclear RNA preparations to conventional RNA‐seq analysis of cellular RNA. The transcript abundance profiles of protein‐coding genes in nuclear RNA and EU‐nuclear RNA were tightly correlated with each other (R² = 0.767), but quite distinct from that of cellular RNA (R² = 0.170 or 0.293). Nuclear and EU‐nuclear RNA reads are frequently mapped across entire genes, including introns, while cellular reads are predominantly mapped to mature transcripts. Both nuclear and EU‐nuclear RNA exhibited a greater ability to detect both protein‐coding and non‐coding expressed genes. 

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3. Bio-orthogonal chemistry enables solid phase synthesis and HPLC and gel-free purification of long RNA oligonucleotides

   https://doi.org/10.1039/d1cc00096a  

   He, Muhan; Wu, Xunshen; Mao, Song; Haruehanroengra, Phensinee; Khan, Irfan; Sheng, Jia; Royzen, Maksim (April 2021, Chemical Communications)

   null (Ed.)

   Solid phase synthesis of RNA oligonucleotides which are over 100-nt in length remains challenging due to the complexity of purification of the target strand from failure sequences. This work describes a non-chromatographic strategy that will enable routine solid phase synthesis of long RNA strands. 

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4. Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

   https://doi.org/10.1093/nar/gkac784  

   Todisco, Marco; Szostak, Jack W. (September 2022, Nucleic Acids Research)

   Abstract Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life. 

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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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