Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS-CoV-2 Replication Complex.

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 (m5 C), N6-methyladenosine (m6 A), N1-methyladenosine (m1 A) and N3-methylcytosine (m3 C) on the activity of SARS-CoV-2 replication complex (SC2RC). We found that Ψ, m5 C, m6 A and m3 C had little effect, whereas m1 A inhibited the enzyme. Both m1 A and m3 C disrupt canonical base pairing, but they had different effects. The fact that m1 A inhibits SC2RC implies that the modification can be difficult to detect. This fact also implies that individuals with upregulated m1 A including cancer, obesity and diabetes patients might have milder symptoms. However, this contradicts clinical observations. Relevant discussions are provided.

Long oligodeoxynucleotides: chemical synthesis, isolation via catching-by-polymerization, verification via sequencing, and gene expression demonstration.

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 in E. 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.

The prevalence of upper respiratory symptoms in a cohort of adults presenting with symptoms of gastro-oesophageal reflux disease.

INTRODUCTION: Gastro-oesophageal reflux disease (GORD) is the pathological reflux of gastric contents into the oesophagus. The oesophagus and the upper respiratory tract have a common origin from the foregut. There is increasing evidence for multiple associations of GORD with the upper respiratory tract. OBJECTIVES: To study the presence of and association of upper respiratory symptoms (URS) with GORD. METHODS: Seventy adults scoring ≥12.5 on a previously validated GORD symptom score (GORD patients) and 70 healthy controls who had infrequent GORD symptoms or no upper gastro-intestinal complaints completed a pre-tested URS questionnaire on the frequency of 14 URS in 5 categories (laryngeal, nasal, pharyngeal, sinusal and aural). All GORD patients underwent upper gastro-intestinal endoscopy. The calculated URS score was correlated against the GORD symptom score and endoscopy findings. RESULTS: URS scores and individual symptom scores were higher in GORD patients compared to controls (mean ± SE, 4.7 ± 4.0; 1.9 ± 2.3). Individuals with higher GORD symptom scores reported more frequent URS. Pharyngeal symptoms had the highest correlation with the GORD symptom score (r=0.507, p<0.001). The presence of oeso-phagitis did not seem to influence the frequency of reporting URS. CONCLUSION: Upper respiratory symptoms are common in individuals with GORD symptoms though there appears to be no association with oesophageal mucosal damage.

Oligonucleotide synthesis under mild deprotection conditions.

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 NaIO4 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 NaIO4 followed by K2CO3. No nucleophilic scavenger such as aniline is needed. Over 10 ODNs including one containing the highly sensitive N4-acetylcytidine were synthesized. Work on extending the method for sensitive RNA synthesis is in progress.