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Abstract This protocol describes the synthesis of long oligonucleotides (up to 401‐mer), their isolation from complex mixtures using the catching‐by‐polymerization (CBP) method, and the selection of error‐free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid‐phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full‐length sequence with a polymerizable tagging phosphoramidite (PTP), co‐polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full‐length sequence from the polymer. Cloning and sequencing guided selection of error‐free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher‐order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Long oligo synthesis Support Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP) Support Protocol 2: Synthesis of 5′‐O‐Bz phosphoramidite Basic Protocol 2: Catching‐by‐polymerization (CBP) purification Basic Protocol 3: Error‐free sequence selection via cloning and sequencing 

Abstract When it is in the template RNA, the naturally occurring m¹A epitranscriptomic RNA modification was recently reported to be able to stop the RNA polymerization reaction catalyzed by the RNA dependent RNA polymerase (RdRp) of SARS-CoV-2. In this report, we report that m¹A via its triphosphate form (m¹ATP) can be incorporated into RNA by the same RdRp. These two findings point a new direction for antiviral drug development based on m¹A for combatting COVID-19. More broadly, it is possible that the large pool of epigenetic RNA as well as DNA modifications could serve as a treasury for drug discovery aimed at combating various infectious and other diseases. 

Abstract This protocol describes a method for the incorporation of sensitive functional groups into oligodeoxynucleotides (ODNs). The nucleophile‐sensitive epigeneticN4‐acetyldeoxycytosine (4acC) DNA modification is used as an example, but other sensitive groups can also be incorporated, e.g., alkyl halide, α‐haloamide, alkyl ester, aryl ester, thioester, and chloropurine groups, all of which are unstable under the basic and nucleophilic deprotection and cleavage conditions used in standard ODN synthesis methods. The method uses a 1,3‐dithian‐2‐yl‐methoxycarbonyl (Dmoc) group that carries a methyl group at the carbon of the methoxy moiety (meDmoc) for the protection of exo‐amines of nucleobases. The growing ODN is anchored to a solid support via a Dmoc linker. With these protecting and linking strategies, ODN deprotection and cleavage are achieved without using any strong bases and nucleophiles. Instead, they can be carried out under nearly neutral non‐nucleophilic oxidative conditions. To increase the length of ODNs that can be synthesized using the meDmoc method, the protocol also describes the synthesis of a PEGylated Dmoc (pDmoc) phosphoramidite. With some of the nucleotides being incorporated with pDmoc‐CE phosphoramidite, the growing ODN on the solid support carries PEG moieties and becomes more soluble, thus enabling longer ODN synthesis. The ODN synthesis method described in this protocol is expected to make many sensitive ODNs that are difficult to synthesize accessible to researchers in multiple areas, such as epigenetics, nanopore sequencing, nucleic acid‐protein interactions, antisense drug development, DNA alkylation carcinogenesis, and DNA nanotechnology. © 2024 Wiley Periodicals LLC. Basic Protocol: Sensitive ODN synthesis Support Protocol 1: Synthesis of meDmoc‐CE phosphoramidites Support Protocol 2: Synthesis of a pDmoc‐CE phosphoramidite 

Abstract SARS‐CoV‐2 causes individualized symptoms. Many reasons have been given. We propose that an individual's epitranscriptomic system could be responsible as well. The viral RNA genome can be subject to epitranscriptomic modifications, which can be different for different individuals, and thus epitranscriptomics can affect many events including RNA replication differently. In this context, we studied the effects of modifications including pseudouridine (Ψ), 5‐methylcytosine (m⁵C),N6‐methyladenosine (m⁶A),N1‐methyladenosine (m¹A) andN3‐methylcytosine (m³C) on the activity of SARS‐CoV‐2 replication complex (SC2RC). We found that Ψ, m⁵C, m⁶A and m³C had little effect, whereas m¹A inhibited the enzyme. Both m¹A and m³C disrupt canonical base pairing, but they had different effects. The fact that m¹A inhibits SC2RC implies that the modification can be difficult to detect. This fact also implies that individuals with upregulated m¹A including cancer, obesity and diabetes patients might have milder symptoms. However, this contradicts clinical observations. Relevant discussions are provided. 

A chemical method suitable for the synthesis of RNAs containing modifications such as N4-acetylcytidine (ac4C) that are unstable under the basic and nucleophilic conditions used by standard RNA synthesis methods is described. The method uses the 4-((t-butyldimethylsilyl)oxy)-2-methoxybutanoyl (SoM) group for the protection of exo-amino groups of nucleobases and the 4-((t-butyldimethylsilyl)oxy)-2-((aminophosphaneyl)oxy)butanoyl (SoA) group as the linker for solid phase synthesis. RNA cleavage and amino deprotection are achieved using fluoride under the same conditions used for the removal of the 2′-OH silyl protecting groups. Using the method, a wide range of electrophilic and base-sensitive groups including those that play structural and regulatory roles in biological systems and those that are artificially designed for various purposes are expected to be able to be incorporated into any position of any RNA sequences. As a proof of concept, a 26-mer RNA containing the highly sensitive ac4C epitranscriptomic modification was successfully synthesized and purified with RP HPLC. MALDI MS analysis indicated that the ac4C modification is completely stable under the fluoride deprotection conditions. The sensitive RNA synthesis method is expected to be able to overcome the long lasting obstacle of accessing various modified sensitive RNAs to projects in areas such as epitranscriptomics, molecular biology and the development of nucleic acid therapeutics. 

The longest oligos that can be chemically synthesized using known methods are typically considered to be 200-mers. Here, we report direct synthesis of an 800-mer green fluorescent protein (GFP) gene and a 1,728-mer Φ29 DNA polymerase gene on an automated synthesizer. Key innovations that enabled the breakthrough include conducting the synthesis on the smooth surface of glass wool or glass bead rather than within the pores of traditional solid supports, and the use of the powerful catching-by-polymerization (CBP) method for the isolation of the full-length oligos from the crude mixture. Conducting the synthesis on smooth surface not only eliminated the steric hindrance that would otherwise prevent long oligo assembly, but also, surprisingly, drastically reduced the errors that commonly occur in traditional oligo synthesis. The long oligos were characterized by cloning followed by Sanger sequencing. We anticipate that the new method for long oligo synthesis will have a significant impact on projects in areas such as synthetic biology, gene editing, protein engineering, and many others. 

The catching-by-polymerization (CBP) oligodeoxynucleotide (oligo or ODN) purification method has been demonstrated suitable for large-scale, parallel, and long oligo purification. The authenticity of the oligos has been verifiedviaDNA sequencing, and gene construction and expression. A remaining obstacle to the practical utility of the CBP method is affordable polymerizable tagging phosphoramidites (PTPs) that are needed for the method. In this article, we report scalable synthesis of the four nucleoside (dA, dC, dG and T) precursors to the PTPs using a route having five steps from inexpensive starting materials. The overall yields ranged from 21% to 35%. The scales of the synthesis presented here are up to 2.1 grams of the precursors. Because the syntheses are chromatography-free, further scaling up the syntheses of the precursors have become more feasible. With the precursors, the PTPs can be synthesized in one step using standard methods involving a chromatography purification. 

Using PEGylated Dmoc (pDmoc) phosphoramidites for oligodeoxynucleotide (ODN) synthesis increases the solubility of ODN on solid support and enables the synthesis of longer sensitive ODNs. 

Over a hundred non-canonical nucleotides have been found in DNA and RNA. Many of them are sensitive toward nucleophiles. Because known oligonucleotide synthesis technologies require nucleophilic conditions for deprotection, currently there is no suitable technology for their synthesis. The recently disclosed method regarding the use of 1,3-dithian-2-yl-methyl (Dim) for phosphate protection and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) for amino protection can solve the problem. With Dim–Dmoc protection, oligodeoxynucleotide (ODN) deprotection can be achieved with NaIO 4 followed by aniline. Some sensitive groups have been determined to be stable under these conditions. Besides serving as a base, aniline also serves as a nucleophilic scavenger, which prevents deprotection side products from reacting with ODN. For this reason, excess aniline is needed. Here, we report the use of alkyl Dim (aDim) and alkyl Dmoc (aDmoc) for ODN synthesis. With aDim–aDmoc protection, deprotection is achieved with NaIO 4 followed by K 2 CO 3 . No nucleophilic scavenger such as aniline is needed. Over 10 ODNs including one containing the highly sensitive N 4 -acetylcytidine were synthesized. Work on extending the method for sensitive RNA synthesis is in progress.