In traditional oligodeoxynucleotide (ODN) synthesis, phosphate groups are protected with the 2-cyanoethyl group, and amino groups are protected with acyl groups. At the end of ODN synthesis, deprotection is achieved with strong bases and nucleophiles. Therefore, traditional technologies are not suitable for the synthesis of ODNs containing sensitive functionalities. To address the problem, we report the use of Dim and Dmoc groups, which are based on the 1,3-dithian-2-yl-methyl function, for phosphate and amine protection for the solid phase ODN synthesis. Using the new Dim–Dmoc protection, deprotection was achieved under mild oxidative conditions without using any strong bases and nucleophiles. As a result, the new technology is suitable for the synthesis of ODNs containing sensitive functions. To demonstrate feasibility, seven 20-mer ODNs including four that contain sensitive ester and alkyl chloride groups were synthesized, purified with RP HPLC, and characterized with MALDI-TOF MS and enzyme digestion essays. High purity ODNs were obtained.
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...
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.
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.
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