Characterization of Byproducts from Chemical Syntheses of Oligonucleotides Containing 1-Methyladenine and 3-Methylcytosine

Abstract

Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact MW by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a MW identical to uncharged m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged m3C in the ∼4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast

“…Mock DNA repair reactions were performed for each DNA substrate in the absence of Tpa1 or AlkB as a positive control for the DNA glycosylase activity of Mag1 and B. subtilis AAG and to reveal the maximum amount of repairable DNA in each initial sample. As noted above, end points for both ϵA-25mer and ϵC-25mer were 0.95, whereas end points for 1mA-25mer and 3mC-25mer substrates were 0.60 (3, 47).…”

“…Different DNA substrates had different end points, which were confirmed in reactions with other glycosylases (data not shown). End points for both ϵA-25mer and ϵC-25mer were 0.95, whereas end points for the more synthetically challenging 1mA-25mer and 3mC-25mer substrates were 0.60 (3, 47). …”

DNA repair enzymes ALKBH2, ALKBH3, and AlkB oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in vitro

“…Ten microgram of AlkB proteins were mixed with 250 ng trypsin in trypsin-ultra buffer, incubating at 37°C overnight. The protein fragments were analyzed by HPLC-MS (24,26). Standard protein fragments (including LFYHGIQPLK and LSLHQDK sequences) were purchased from New England Peptide (MA).…”

Hydrolyzable Tannins Are Iron Chelators That Inhibit DNA Repair Enzyme ALKBH2

“…To test the inhibitory effects of hydrolyzable tannins on ALKBH2, 16mer oligonucleotides bearing a site-specific m3C/m1A adduct, the primary substrate of ALKBH2, were synthesized by using solid-phase phosphoramidite chemistry. 12,15 The recombinant human ALKBH2 protein was expressed and purified according to previous studies. 13,14 Then, the repair efficiency of ALKBH2 on m3C/m1A was assessed.…”