A New Method for the Postsynthetic Generation of Abasic Sites in Oligomeric DNA

Abstract

The abasic site is the most common lesion in DNA and is thought to play a critical role in mutagenesis. However, a general chemical method for the site-specific generation of true abasic sites in oligodeoxynucleotides has been lacking. We now describe a procedure which permits the postsynthetic generation of abasic sites in single- or double-stranded DNA oligomers without regard to their base composition. An appropriately protected 3-deoxyhexitol was synthesized and used as the monomer that was incorporated into DNA oligomers using the standard phosphoramidite method for automated DNA synthesis. The resulting stable diol-containing oligonucleotides were purified by HPLC and converted quantitatively into the corresponding abasic DNA sequences by mild periodate oxidation. The abasic site in DNA was found to be relatively stable at room temperature, but was completely cleaved when treated with putrescine at 95 degrees C. Identification of the major degradation products was accomplished by gel electrophoresis, HPLC isolation, and characterization by electrospray ionization mass spectrometry. The thermal stabilities of duplex oligonucleotides containing a natural abasic site were studied, and the results were compared with those from oligomers containing T/dA, F/dA, or the precursor diol opposite dA at the same site.

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

“…By using both MALDI and ESI, we confirmed the molecular weight (to within Ϯ0.5 u for ESI and Ϯ1.0 u for MALDI) for all the ODNs containing an abasic site (Table 1). We found the abasic species to be relatively stable for these experiments, in agreement with Shishkina and Johnson [28].…”

Bioanalytical LC–MS of Therapeutic Oligonucleotides

“…n Nonspecific binding to columns, containers & glass ware pipette tips, for example Several authors reported that the exposed silanol groups on reversed-phase C 18 columns can irreversibly adsorb OGNTs [45,91,92], leading to poor assay sensitivity, reproducibility and carry-over [45]. Particle size, column length, mobile phase, flow rate and separation temperature were found to influence resolution and sensitivity of OGNTs [63].…”

“…For RafAON, stability was determined in plasma during long-term storage (monkey: 62 days at -70°C; human: 61 days at -20°C), during freezethaw (two cycles in mouse/monkey, three cycles in human plasma), at room temperature (1.5 h for human but not in mouse/monkey plasma) thawed on ice for up to 2 h (mouse/monkey), and in processed samples when stored at 4°C (mouse/ monkey: 24 h and human plasma: 51 h [28,68]). Shishkina and Johnson reported that OGNTs are susceptible to degradation during evaporation to dryness [92]. The elution solvent is very important for stabilizing OGNT during evaporation.…”

“…The elution solvent is very important for stabilizing OGNT during evaporation. The use of a high-percentage of MeCN in TEA buffer, in combination with evaporation to 'almost' dry (2-3 µl), appears to stabilize the OGNT during sample preparation [92].…”

Site Specific Synthesis and Polymerase Bypass of Oligonucleotides Containing a 6-Hydroxy-3,5,6,7-tetrahydro-9H-imidazo[1,2-a]purin-9-one Base, an Intermediate in the Formation of 1,N2-Etheno-2‘-deoxyguanosine

“…Periodate oxidation of a vicinal diol is a common strategy for the incorporation of aldehyde functionality into oligonucleotides under mild and neutral conditions (35)(36)(37)(43)(44)(45)(46)(47)(48)(49)(50)(51). dGuo was alkylated at the N1-position with allyl bromide under alkaline conditions to give the N1-propenyl derivative (13) in 66% yield (Scheme 3) (34,43,52,53).…”