Study on steady-state kinetics of nucleotide analogues incorporation by non-gel CE.

A method for studying steady-state kinetics of nucleotide analogues incorporation into DNA strand by non-gel CE (NGCE) with LIF was developed. Nucleoside analogue is a kind of antiviral agent used to inhibit viral replication in infected cells, especially HIV. Steady-state parameter K(m) for nucleotide analogues is determined to imply the relationship between nucleoside analogues and the enzyme in the DNA chain elongation and predict the antiviral efficacy in vivo. Samples were prepared by single nucleotide incorporation assays catalyzed by Taq DNA polymerase at 58 degrees C and HIV reverse transcriptase (RT) at 37 degrees C, and then were separated using NGCE under optimized conditions: 25 mmol/L Tris-boric-EDTA buffer (pH 8.0) with 7 mmol/L urea in the presence of 20% w/v PEG 35000 at 30 degrees C and -20 kV. K(m(dTTP)), K(m(d4TTP)) and K(m(AZTTP)) were measured by NGCE for the first time and their values for Taq DNA polymerase were 0.29+/-0.04, 32.1+/-3.3 and 74.5+/-6.6 micromol/L, respectively. For HIV RT, the values were 0.15+/-0.05, 0.31+/-0.03 and 0.17+/-0.03 micromol/L, respectively. The trend of data for HIV RT measured by NGCE was consistent with that measured by PAGE. The reported method by NGCE for the K(m) determination was powerful, sensitive and fast, and required less amounts of reagents compared with PAGE. It be employed as a reliable alternative method and further applied in other relative studies of nucleoside analogue substrates and DNA polymerases or RTs.

Synthesis and recognition of novel isonucleoside triphosphates by DNA polymerases.

Isonucleosides have been attracting a lot of attention in recent years due to the chemical and enzymatic stability and potential anticancer and antiviral activities. We have reported some of the isonucleosides which exhibited significant anticancer activity and found that the oligonucleotide incorporated with isonucleoside could increase the enzymatic stability against the degradation by phosphodiesterase. In this paper, we investigated the recognition of the isonucleoside triphosphates 1-6 by Taq, Vent(exo(-)), DeepVent(exo(-)), 9 degrees Nm, and Therminator DNA polymerases by a non-radioactivity method. We found that most of the isonucleoside triphosphates can be recognized by various DNA polymerase and act as terminators. Isonucleoside triphosphates 2 and 6 can be incorporated as substrates into the primer at 3' terminus to lengthen the chain dependent on a DNA template by Vent(exo(-)) and DeepVent(exo(-)) DNA polymerases.