Oligodeoxynucleotides (ODNs) are typically purified and analysed with HPLC equipped with a UV-Vis detector. Quantities of ODNs are usually determined using a UV-Vis spectrometer separately after HPLC, and are reported as optical density at 260 nm (OD260). Here, we describe a method for direct determination of OD260 of ODNs using the area of the peaks in HPLC profiles. It is expected that the method will save significant time for researchers in the area of nucleic acid research, and minimize the loss of oligonucleotide samples.
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, the modifications 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), 5methylcytosine (m 5 C), N 6 -methyladenosine (m 6 A), N 1 -methyladenosine (m 1 A) and N 3methylcytosine (m 3 C) on the activity of SARS-CoV-2 replication complex (SC2RC). We found that Ψ, m 5 C, m 6 A and m 3 C had little effects, while m 1 A inhibited the enzyme. Both m 1 A and m 3 C disrupt canonical base-pairing, but they had different effects. The fact that m 1 A inhibits SC2RC implies that the modification can be difficult to detect. The fact also implies that individuals with upregulated m 1 A including cancer, obesity and diabetes patients may have milder symptoms. However, this contradicts clinical observations. Relevant discussions are provided.
Like other viruses, SARS-CoV-2 causes different symptoms and different degrees of harmfulness to different individuals. Potential reasons include an individual’s viral dose exposure, the affinity of an individual’s ACE2 to the spike protein of the virus, and the ability of the individual’s induced immune system to neutralize the virus. Beyond these, an individual’s epitranscriptomic system could be among the causes as well. The viral RNA genome, once inside the host cell, can be subject to modifications by the host’s epitranscriptomic machinery. Because the machinery is different in different individuals, it is reasonable to believe that RNA modifications are different among different individuals, and can positively or negatively affect downstream events that involve the RNA such as replication of viral genome, generation of viral mRNAs, viral protein production, RNA recognition by host’s immune system, and packaging of RNA genome into new viral particles. In this context, we studied the effects of several RNA modifications including pseudouridine (Ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A), N1-methyladenosine (m1A) and N3-methylcytosine (m3C) on the catalytic activity of SARS-CoV-2 replication complex (SC2RC), which included RNA dependent RNA polymerase (RdRp). We found that Ψ, m5C, m6A and m3C had little effects on the activity, while m1A severely inhibited the enzyme. Both m1A and m3C disrupt canonical base pairing. It is interesting one of them inhibits the enzyme while the other does not. The fact that m1A inhibits SC2RC may imply that the modification can be difficult to identify using any method even though it may exist and play a critical role. Putting aside other mechanisms by which the modifications cause individualized symptoms, the results indicated that individuals with a higher chance of m1A modification may stop viral replication and have less severe symptoms. However, this contradicts the observations that individuals with clinical conditions such as cancer, obesity and diabetes, who have upregulated m1A modifications, are more vulnerable to COVID-19. This contradiction may be explained by the importance of the dynamic nature of epitranscriptomic modifications for viral survival.
Like other viruses, SARS-CoV-2 causes different symptoms and different degrees of harmfulness to different individuals. Potential reasons include an individual’s viral dose exposure, the affinity of an individual’s ACE2 to the spike protein of the virus, and the ability of the individual’s induced immune system to neutralize the virus. Beyond these, an individual’s epitranscriptomic system could be among the causes as well. The viral RNA genome, once inside the host cell, can be subject to modifications by the host’s epitranscriptomic machinery. Because the machinery is different in different individuals, it is reasonable to believe that RNA modifications are different among different individuals, and can positively or negatively affect downstream events that involve the RNA such as replication of viral genome, generation of viral mRNAs, viral protein production, RNA recognition by host’s immune system, and packaging of RNA genome into new viral particles. In this context, we studied the effects of several RNA modifications including pseudouridine (Ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A), N1-methyladenosine (m1A) and N3-methylcytosine (m3C) on the catalytic activity of SARS-CoV-2 replication complex (SC2RC), which included RNA dependent RNA polymerase (RdRp). We found that Ψ, m5C, m6A and m3C had little effects on the activity, while m1A severely inhibited the enzyme. Both m1A and m3C disrupt canonical base pairing. It is interesting one of them inhibits the enzyme while the other does not. The fact that m1A inhibits SC2RC may imply that the modification can be difficult to identify using any method even though it may exist and play a critical role. Putting aside other mechanisms by which the modifications cause individualized symptoms, the results indicated that individuals with a higher chance of m1A modification may stop viral replication and have less severe symptoms. However, this contradicts the observations that individuals with clinical conditions such as cancer, obesity and diabetes, who have upregulated m1A modifications, are more vulnerable to COVID-19. This contradiction may be explained by the importance of the dynamic nature of epitranscriptomic modifications for viral survival.
Over a hundred non-canonical nucleotides have been found in DNA and RNA. Many of these modified nucleotides are sensitive toward nucleophiles and bases. Because known solid phase DNA and RNA synthesis technologies require strongly nucleophilic and basic conditions for deprotection and cleavage, there is no technology for the synthesis of DNAs and RNAs containing these sensitive nucleotides. The Dim-Dmoc technology has been developed to overcome the challenge. With Dim-Dmoc protection, oligodeoxynucleotide (ODN) deprotection has be achieved with sodium periodate oxidation followed by β-elimination induced by the weak base aniline. Some sensitive groups have been shown to be stable under the deprotection conditions. Besides serving as a base, aniline also serves as a nucleophilic scavenger for the side products of Dim-Dmoc deprotection, which prevents the side products from reacting with the deprotected ODN. For this reason, excess aniline is needed. In this article, we report the use of alkyl Dim (aDim) and alkyl Dmoc (aDmoc) protecting groups for ODN synthesis. With aDim-aDmoc protection, deprotection is demonstrated to be achievable with sodium periodate oxidation followed by the non-nucleophilic base potassium carbonate at pH 8. No scavenger for the side products of deprotection is needed. Over 10 ODNs with length ranging from 10-mer to 23-mer were synthesized, and importantly, the ODNs could be easily purified with RP HPLC. It was further demonstrated that the highly sensitive N4-acetylcytidine nucleoside could survive the oxidative deprotection conditions, and ODNs containing this sensitive nucleotide could be readily synthesized and purified without the need of any special cautions. Work on extending the method for the synthesis of sensitive RNAs such as those containing the biologically important ac4C is in progress.
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