2'-Fluoro modified nucleic acids: Polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry

Abstract

Fragmentation is a major factor limiting mass range and resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protonation of the nucleobase leads to base loss and backbone cleavage by a mechanism similar to the depurination reactions employed in the chemical degradation method of DNA sequencing. In a previous study, the stabilizing effect of substituting the 2' hydrogen with an electronegative group such as hydroxyl or fluorine was investigated. These 2' substitutions stabilized the N-glycosidic linkage, blocking base loss and subsequent backbone cleavage. For such chemical modifications to be of practical significance, it would be useful to be able to employ the corresponding 2'-modified nucleoside triphosphates in the polymerase-directed synthesis of DNA. This would provide an avenue to the preparation of 2'-modified PCR fragments and dideoxy sequencing ladders stabilized for MALDI analysis. In this paper methods are described for the polymerase-directed synthesis of 2'-fluoro modified DNA, using commercially available 2'-fluoronucleoside triphosphates. The ability of a number of DNA and RNA polymerases to incorporate the 2'-fluoro analogs was tested. Four thermostable DNA polymerases [Pfu (exo-), Vent (exo-), Deep Vent (exo-) and UITma] were found that were able to incorporate 2'-fluoronucleotides with reasonable efficiency. In order to perform Sanger sequencing reactions, the enzymes' ability to incorporate dideoxy terminators in conjunction with the 2'-fluoronucleotides was evaluated. UITma DNA polymerase was found to be the best of the enzymes tested for this purpose. MALDI analysis of enzymatically produced 2'-fluoro modified DNA using the matrix 2,5-dihydroxy benzoic acid showed no base loss or backbone fragmentation, in contrast to the extensive fragmentation evident with unmodified DNA of the same sequence.