2'-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry.
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 [Tang,W., Zhu,L. and Smith,L.M. (1997) Anal. Chem ., 69, 302-312], the stabilizing effect of substituting the 24 hydrogen with an electronegative group such as hydroxyl or fluorine was investigated. These 24 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 24-modified nucleoside triphosphates in the polymerase-directed synthesis of DNA. This would provide an avenue to the preparation of 24-modified PCR fragments and dideoxy sequencing ladders stabilized for MALDI analysis. In this paper methods are described for the polymerase-directed synthesis of 24-fluoro modified DNA, using commercially available 24-fluoronucleoside triphosphates. The ability of a number of DNA and RNA polymerases to incorporate the 24-fluoro analogs was tested. Four thermostable DNA polymerases [Pfu (exo-), Vent (exo-), Deep Vent (exo-) and UlTma] were found that were able to incorporate 24-fluoronucleotides with reasonable efficiency. In order to perform Sanger sequencing reactions, the enzymes' ability to incorporate dideoxy terminators in conjunction with the 24-fluoronucleotides was evaluated. UlTma DNA polymerase was found to be the best of the enzymes tested for this purpose. MALDI analysis of enzymatically produced 24-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.
Ono
T
T
Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Scalf
M
M
Smith
L M
LM
D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D054306:Dideoxynucleotides; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D017611:Fluorine Compounds / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013942:Thymine Nucleotides
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D017611:Fluorine Compounds; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; C024107:2'-deoxycytidine 5'-triphosphate; C025664:2'-fluoro-2'-deoxyadenosine triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D005092:Exonucleases; D008274:Magnesium; C017216:2',3'-dideoxythymidine triphosphate