Dissolve‐spin: Desalting oligonucleotides for MALDI MS analysis

Apostle, Alexander; Fang, Shiyue

doi:10.1002/jms.4893

Cited by 3 publications

(2 citation statements)

References 28 publications

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“…All RNAs except for 1a, which was purchased from a commercial source, were synthesized using the phosphoramidite chemistry, purified with RP HPLC and characterized with MALDI MS following reported procedures. [11][12][13][14] The phosphoramidite monomers for the synthesis were from commercial sources except for the m 3 C phosphoramidite monomer, which was synthesized in consultation of reported procedures 11,15 with modifications (supporting information). RNA 1a had the fluorophore FAM at its 5'-end, which was intended to provide an additional means for the analysis of the results of the extension reactions.…”

Section: Resultsmentioning

confidence: 99%

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Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS‐CoV‐2 Replication Complex**

et al. 2023

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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.

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Section: Resultsmentioning

confidence: 99%

“…The solution was evaporated to dryness and analyzed with RP HPLC using conditions described elsewhere [28] . All RNAs were desalted using RP HPLC or the dissolve‐spin method [6d] and characterized using MALDI MS.…”

Section: Methodsmentioning

confidence: 99%

Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS‐CoV‐2 Replication Complex**

et al. 2023

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Advances in nucleic acid sample preparation for electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry

Sharin,

Floro,

Clark

2023

International Journal of Mass Spectrometry

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

Apostle

Yin

Chillar

et al. 2022

Preprint

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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.

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Dissolve‐spin: Desalting oligonucleotides for MALDI MS analysis

Cited by 3 publications

References 28 publications

Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS‐CoV‐2 Replication Complex**

Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS‐CoV‐2 Replication Complex**

Advances in nucleic acid sample preparation for electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry

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

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