Quantitative measurements on the duplex stability of 2,6-diaminopurine and 5-chloro-uracil nucleotides using enzymatically synthesized oligomers.
2,6-Diaminopurine and 5-chloro-uracil 2'-deoxynucleoside 5'-triphosphates were synthesized from their 2'-deoxynucleosides. Using a method of creating oligonucleotides by enzymatic primer extension, dodecanucleotides representing an XbaI/SalI site and the complementary SalI/XbaI site were generated containing these base modifications. Their duplex stability was quantitatively compared by thin-layer chromatography to oligomers containing 2'-deoxyadenosine and 2'-deoxythymidine. The two unmodified oligomers already showed significant differences in dissociation temperature and binding equilibrium. Substitution with 5-chloro-2'-deoxyuridine did not affect the dissociation temperature of either oligomer, the 2,6-diaminopurine, however, led to an increase of 1.8 degrees C or 1.5 degrees C per modified base, respectively. While in the XbaI/SalI oligomer both base modifications changed the binding equilibrium, the 2,6-diaminopurine by a factor of 1.32, the 5-chloro-uracil by 0.65, no such effect was found with the complementary oligomer.
Hoheisel
J D
JD
Imperial Cancer Research Fund, London, UK.
Lehrach
H
H
D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D004355:Drug Stability; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D010766:Phosphorylation; D015183:Restriction Mapping; D013816:Thermodynamics
D003838:Deoxyadenine Nucleotides; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; C066589:5-chloro-2'-deoxyuridine 5'-triphosphate; C033846:2-amino-2'-deoxyadenosine 5'-triphosphate