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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 

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. 

Abstract In Arabidopsis (Arabidopsis thaliana), overproduction of salicylic acid (SA) increases disease resistance and abiotic stress tolerance but penalizes growth. This growth–defense trade-off has hindered the adoption of SA-based disease management strategies in agriculture. However, investigation of how SA inhibits plant growth has been challenging because many SA-hyperaccumulating Arabidopsis mutants have developmental defects due to the pleiotropic effects of the underlying genes. Here, we heterologously expressed a bacterial SA synthase gene in Arabidopsis and observed that elevated SA levels decreased plant growth and reduced the expression of cold-regulated (COR) genes in a dose-dependent manner. Growth suppression was exacerbated at below-ambient temperatures. Severing the SA-responsiveness of individual COR genes was sufficient to overcome the growth inhibition caused by elevated SA at ambient and below-ambient temperatures while preserving disease- and abiotic-stress-related benefits. Our results show the potential of decoupling SA-mediated growth and defense trade-offs for improving crop productivity. 

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. 

Long oligodeoxynucleotides (ODNs) are segments of DNAs having over one hundred nucleotides (nt). They are typically assembled using enzymatic methods such as PCR and ligation from shorter 20 to 60 nt ODNs produced by automated de novo chemical synthesis. While these methods have made many projects in areas such as synthetic biology and protein engineering possible, they have various drawbacks. For example, they cannot produce genes and genomes with long repeats and have difficulty to produce sequences containing stable secondary structures. Here, we report a direct de novo chemical synthesis of 400 nt ODNs, and their isolation from the complex reaction mixture using the catching-by-polymerization (CBP) method. To determine the authenticity of the ODNs, 399 and 401 nt ODNs were synthesized and purified with CBP. The two were joined together using Gibson assembly to give the 800 nt green fluorescent protein (GFP) gene construct. The sequence of the construct was verified via Sanger sequencing. To demonstrate the potential use of the long ODN synthesis method, the GFP gene was expressed inE. coli. The long ODN synthesis and isolation method presented here provides a pathway to the production of genes and genomes containing long repeats or stable secondary structures that cannot be produced or are highly challenging to produce using existing technologies. 

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.