EDUCACIÓN PRIMARIA INDÍGENA
4.1 Reconoce que la función docente debe ser ejercida con apego a los fundamentos legales, los principios
Scheme 6.2 Synthesis of S-mercaptopropyl-5-(mercaptomethyl)-2'-deoxyuridine-5'-triphosphate, HSTTP (10).
6.3.2 HPLC Purification and Analysis
The crude AcSTTP (9)was purified and analyzed by RP-HPLC. The AcSTTP was eluted at 260
nm, with a flow-rate of 6 mL/min [buffer A: 20 mM TEAAc (pH 7.1) in water; buffer B: 20 mM TEAAc (pH7.1) in 50% acetonitrile] and a linear gradient from 5 % buffer B to 40 % buffer B in 20 min. The desired peak was collected and the buffer was removed by lyophilization. All the dry frac- tions of AcSTTP and precipitated HSTTP were analyzed on Welchrom C18-XB column (4.6*250 mm)
and measured at a flow rate of 1.0 mL/min and a linear gradient of 0 to 25 % buffer B in 20 min [buffer A: 20 mM TEAAc (pH 7.1) in water; buffer B: 20 mM TEAAc (pH 7.1) in 50% acetonitrile (figure 5.1). The compounds being structurally similar have a small, but measureable, difference in
their retention times on the column. A RP-HPLC analysis of purified AcSTTP and HSTTP, at 260 nm
wavelength, is presented in figure 6.1.
Figure 6.1 RP-HPLC analysis of AcSTTP and HSTTP: HSTTP (red curve, retention time: 24.75 min); AcSSTTP (blue curve, reten-
tion time: 24.96 min) and co-injection of AcSTTP and HSTTP (black curve, retention times: 24.75 and 24.96 min, respective-
ly).
6.3.3 Absorption Properties
The native thymidine-5'-triphosphate (TTP) shows an absorption maximum at ~267 nm. The modification at the C5-methyl of the thymine base does not alter the absorption properties of the resultant nucleobase. Hence, S-(3(acetylthio)propyl)-5-(mercaptomethyl)-2'-deoxyuridine-5'- triphosphate (AcSTTP, 9) and S-mercaptopropyl-5-(mercaptomethyl)-2'-deoxyuridine-5'-
.
Figure 6.2 Absorption spectra of TTP (—), AcSTTP (—), and HSTTP (—) in water at 25 ˚C.
6.3.4 Enzymatic Incorporation of TTP, AcSTTP and HSTTP into DNA
To investigate the incorporation efficiency of S-(3(acetylthio)propyl)-5-(mercaptomethyl)- 2'-deoxyuridine-5'-triphosphate (AcSTTP, 9) and S-mercaptopropyl-5-(mercaptomethyl)-2'-
deoxyuridine-5'-triphosphate, (HSTTP,10) into DNA, polymerization reactions were performed in
the presence of klenow exo minus DNA polymerase (Kf-). A list of DNA primers and templates used for the primer extension reactions is presented in table 6.1.
Table 6.1 Sequences of DNA primers and templates used for the enzymatic incorporation of TTP, AcSTTP and HSTTP. The
underlined bases are the sites for incorporation of TTP, AcSTTP and HSTTP.
Primers: P1 (17-mer) 5'-d-TAG CGG GTT GCT GGT GG -3' P2 (21-mer) 5'-d-GCG TAA TAC GAC TCA CTA TAG-3' Templates:T1 (21-mer) 3'-d-ATC GCC CAA CGA CCA CCA TGG- 5'
T2 (23-mer) 3'-d-ATC GCC CAA CGA CCA CCAAAT GG- 5'
Figure 6.3 Enzymatic incorporation of a single nucleotide (TTP, AcSTTP and HSTTP) into DNA (T1): (a) Enzymatic incorpo-
ration of TTP, AcSTTP and HSTTP by Klenow exo(-) on DNA template T1. Gel electrophoresis autoradiography of the
polymerization reaction with (b) Kf-= 0.0015 U/µL and reaction time = 60 min for TTP and 90 min for AcSTTP and HSTTP,
(c) Kf-= 0.00075 U/µL and reaction time = 60 min, (d) Kf-= 0.0015 U/µL and reaction time = 30 min, and (d) Kf-= 0.00075 U/µL and reaction time = 30 min.
The DNA polymerization was performed with primer (1.5 μM), template (3 μM), TTP/
AcSTTP/ HSTTP (0.1 mM), and Klenow exo(-) DNA polymerase (varying amounts) and analyzed by
19% polyacrylamide gel electrophoresis (figures 6.3, 6.4 and 6.5). Like TTP, AcSTTP and HSTTP also
act as fine substrates for DNA polymerases. Single nucleotide incorporation of the TTP, AcSTTP and HSTTP into DNA was studied using a short, 21 nt long DNA template (T1) and the results are shown
by TTP and HSTTP: under different reaction times and with different enzyme concentrations, AcSTTP
always terminates polymerization after the incorporation of a single nucleotide (n + 1 DNA, figure 6.3, lane 4) whereas TTP and HSTTP either result in the formation of n + 2 DNA product or a mixture
of desired n + 1 DNA along with some n + 2 DNA (figure 6.3, lanes 2 and 3, respectively). From the intensity of the product DNA band it appears that both, AcSTTP and HSTTP, are well recognized by the
DNA polymerase. With the modified DNA moving slower than the native DNA, there is a measure- able structural impact of the modification on the product mobility. The single nucleotide incorpora- tion will be further confirmed by MALDI-MS analysis.
The DNA template T2 was designed to study the efficiency of incorporation of three consec- utive modified bases. The 17 nt long primer P1 was enzymatically extended to a 20 nt DNA product by incorporating three consective thymidine bases over the template T2. The DNA polymerization was performed with primer (1.5 μM), template (3 μM), TTP/ AcSTTP/ HSTTP (0.1 mM), and Klenow
exo(-) DNA polymerase (0.0015 U/µL) and analyzed by 19% polyacrylamide gel electrophoresis (figure 6.4). The reaction was incubated for 5 min with TTP as a substrate (figure 6.4, lane 2) whereas the reactions with AcSTTP and HSTTP as substrates, the incubation time was 60 min (figure
6.4, lanes 3 and 4, respectively). Under these experimental conditions, AcSTTP appears to be the best
substrate for DNA polymerase.
We also studied the efficiency of incorporation using a longer DNA template (T3). The in- corporation was carried out by extending a 21 nucleotide primer along a 55 nucleotide template. The DNA polymerization was performed with primer (1.5 μM), template (5 μM), TTP/ AcSTTP/ HSTTP and other dNTPs (0.1 mM), and Klenow exo(-) DNA polymerase (0.04 U/μL) and analyzed by
19% polyacrylamide gel electrophoresis. Figure 3.8 shows the primer extension using T2 template. This polymerization involved the incorporation of three TTP/ AcSTTP/ HSTTP (figure 6.5).
Figure 6.4 Enzymatic incorporation of three consecutive bases (TTP, AcSTTP and HSTTP) into DNA (T2): (a) Enzymatic
incorporation of TTP, AcSTTP and HSTTP by Klenow exo(-) on DNA template T2. (b) Gel electrophoresis autoradiography of
the polymerization reaction with Kf-= 0.0015 U/µL and reaction time = 5 min for TTP (lane 2) and 60 min for AcSTTP (lane
4) and HSTTP (lane 3).
Figure 6.5 Enzymatic incorporation of all natural dNTPs, TTP, AcSTTP and HSTTP into DNA (T3): a) Enzymatic incorpora-
tion of all dNTPS, TTP, AcSTTP and HSTTP by Klenow exo(-) on DNA template T3;(b) Gel electrophoresis autoradiography
of the polymerization reaction with Kf- = 0.0015 U/µL and reaction time = 60 min for TTP (lane 3) and 90 min for AcSTTP
6.3.5 Other Proposed Studies
The synthesized and purifiedAcST-DNA and HST-DNA will be used to study the stabilization of
gold nanoparticles with reference to the native DNA. The study will be based on the strong affinity of gold nanoparticles for thiol-functionality. HST-DNA will be used as such to reduce gold whereas AcST-DNA will be hydrolyzed in-situ with ammonia to generate free thiol-group for gold nanoparti-
cle stabilization. The modified DNA will also find use and application in DNA/RNA microchip sur- face functionalization for rapid detection of pathogens.
6.4Conclusions and Future Prospects
We have successfully synthesized a novel nucleoside, S-(3(acetylthio)propyl)-5- (mercaptomethyl)-2'-deoxyuridine, from thymidine. This modified nucleoside was successfully used to synthesize S-(3(acetylthio)propyl)-5-(mercaptomethyl)-2'-deoxyuridine-5'-triphosphate and S-mercaptopropyl-5-(mercaptomethyl)-2'-deoxyuridine-5'-triphosphate. Both the forms of the triphosphates are efficient substrates for DNA polymerases. The polymerase recognition and incor- poration efficiency of the modified triphosphates, relative to the native, has also been effectively demonstrated by the use of different combination of DNA primers and templates. The modified DNA thus obtained will be further used for gold nanoparticle stabilization. The modified DNA will also find use and application in DNA/RNA microchip surface functionalization for rapid detection of pathogens.
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