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Segmented hybridization probes: modulating target affinity and base pairing selectivity

100%

Egetenmeyer S., Geiger E., Richert C.

BioTechnologia. Journal of Biotechnology Computational Biology and Bionanotechnology

2012

tom 93

nr 3

Template-directed base pairing of 2-chloro-2'-deoxyadenosine catalyzed by AMV reverse transcriptase

84%

Bukowska-Maciejewska A. M., Kusmierek J. T.

Acta Biochimica Polonica

1998

tom 45

nr 2

2-Chloro-2'-deoxyadenine (2CldA) is used for treatment of several lymphoid malignancies. Since this drug is incorporated into DNA, we have undertaken studies on base pairing of 2-chloroadenine (2ClA). 2CldA phosphoramidite was synthesized and used for preparation of 25-mer templates with 2ClA located at site 21 from the 3'-end. Kinetic parameters (Km and Vmax) for the incorporation of deoxynucleoside-5'-triphosphates by AMV reverse transcriptase opposite the 2ClA template, as well as for the extension of 2ClA·T pair, were determined. The efficiency (Vmax/Km) of incorporation of dGTP, dCTP, and dATP opposite 2ClA is at least one order of magnitude lower than opposite unmodified A. The efficiency of incorporation of dTTP opposite 2ClA is about 30-fold lower than opposite A and extension of 2ClA·T pair is 3-fold lower than of A·T pair. From the analysis of the parameters of dTTP incorporation we conclude that formation of 2ClA·T pair is thermodynamically, but not kinetically controlled. The difference in binding energy (ΔΔG) between 2ClA·T and A·T pairs in the environment of the polymerase active site is 2 kcal/mol. Our results indicate that the presence of 2ClA in DNA slows down replication, but does not lead to base-substitution mutations.

Ograniczanie wyników

1 Acta Biochimica Polonica

1 BioTechnologia. Journal of Biotechnology Computational Biology and Bionanotechnology

1 Bukowska-Maciejewska A. M.

1 Egetenmeyer S.

1 Geiger E.

1 Kusmierek J. T.

1 Richert C.

1 2012

1 1998

Wyniki wyszukiwania

Segmented hybridization probes: modulating target affinity and base pairing selectivity

Template-directed base pairing of 2-chloro-2'-deoxyadenosine catalyzed by AMV reverse transcriptase