Synthesis and DNA-polymerase incorporation of colored 4-selenothymidine triphosphate for polymerase recognition and DNA visualization

Article abstract of DOI:10.1002/anie.200705213

Highlighting changes in yellow: The replacement of a single oxygen atom in thymidine triphosphate with a selenium atom gave yellow 4-selenothymidine 5′-triphosphate (^SeTTP; see solutions of colorless natural TTP (left) and ^SeTTP (right)). ^SeTTP is recognized by DNA polymerase. Its incorporation into DNA (see scheme) yields colored DNA and occurs with the same level of efficiency as the incorporation of natural TTP. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200705213

Angewandte
Chemie
DOI: 10.1002/anie.200705213
DNA Modification
Synthesis and DNA-Polymerase Incorporation of Colored
4-Selenothymidine Triphosphate for Polymerase Recognition and
DNAVisualization**
Julianne Caton-Williams and Zhen Huang*
DNA polymerase replicates cellular DNA in a template-
dependent manner by using deoxyribonucleotide triphos-
phates (dNTPs). Specific recognition occurs through base
pairing and size-and-shape matching.[1] Extensive studies
have focused on the recognition and stability of the DNA
double-stranded structure and on the interaction between the
dNTP substrate, the template, and DNA polymerase.[2] In the
chemogenetic investigation of the structure and function of
nucleic acids, we pioneered the atom-specific substitution of
an oxygen atom in the nucleic acid (atomic radius: 0.73 Š)
with selenium (1.16 Š), which is also in Group 16 of the
periodic table, as an atomic probe.[3] Our recent replacement
Figure 1. UV spectra of TTP (red, lmax =267 nm) and SeTTP (gray,
of the oxygen atom at the 4-position of thymidine with a
selenium atom revealed that the DNA duplex is flexible
lmax =369 nm). Inset: left: TTP (colorless); right: SeTTP (yellow).
enough to accommodate the large selenium atom and that the
[4]
=
C Se functionality is relatively stable. Furthermore, we
the development of novel DNA probes for visualization
without structure perturbation.
discovered that the 4-Se atom forms a selenium-mediated
À
hydrogen bond with the 6-amino group of adenosine (Se···H
Our previous attempts to synthesize SeTTP failed as a
result of deselenization. Following the development of a
useful protecting group for Se,[12] we finally completed the
synthesis of SeTTP (Scheme 1). This innovative strategy
enables both the incorporation of Se and the protection of
2. After the removal of the 5’-DMTr and 3’-TMS protecting
N), and that the crystal structure of the DNA molecule with
the Se-modified nucleobase is virtually identical to that of the
corresponding native DNA.[4]
We have now discovered colored 4-Se-thymidine 5’-
triphosphate (SeTTP; Figure 1). We formed yellow SeTTP
from colorless native TTP by altering a single atom and
observed the recognition and incorporation of SeTTP by DNA
polymerase. The visualization of DNA is of interest and
importance for many biochemical, biological, and medical
applications,[5–7] such as gene-expression analysis and the
detection of human diseases and pathogens. DNA is usually
visualized by using intercalated dye molecules[8,9] and specific
DNA probes tagged with fluorescent moieties.[5,10,11] How-
ever, these often bulky intercalated dye molecules and
tethered fluorescent moieties can cause the perturbation of
DNA duplex structures. We describe herein the synthesis of
colored SeTTP and its efficient incorporation into DNA
molecules by DNA polymerase. This method should lead to
groups, the 5’-OH group of 3 was phosphorylated by treat-
[13]
ment with POCl3
followed by pyrophosphate, and the
cyanoethyl protecting group was removed under mild con-
ditions by treatment with K2CO3. The cyanoethyl group may
also be removed prior to the phosphorylation step.
[*] J. Caton-Williams, Prof. Dr. Z. Huang
Department of Chemistry
Georgia State University
Atlanta, GA 30303 (USA)
Fax: ( +1)404-413-5505
E-mail: huang@gsu.edu
Scheme 1. Chemical synthesis of SeTTP (4) and enzymatic synthesis of
SeT-containing DNA (5): a) N-trimethylsilylimidazole; 1,2,4-triazole,
POCl3, Et3N; b) (NCCH2CH2Se)2, NaBH4, EtOH; c) 80% acetic acid;
d) POCl3, Me3PO4; tri-n-butylamine, pyrophosphate, N,N-dimethylform-
amide; H2O; e) aqueous K2CO3 (0.05m); f) template-dependent DNA
polymerization. DMTr=4,4’-dimethoxytrityl, TMS=trimethylsilyl.
[**] This research was supported by GSU and the U.S. National Science
Foundation (MCB-0517092).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
The Se-modified triphosphate was purified by HPLC, and
its identity was confirmed by HPLC and HRMS (see the
Supporting Information). We found that the selenium func-
tionality of SeTTP is stable in air under aqueous conditions.
The UV spectrum of SeTTP (lmax = 369 nm, e = 1.8 ”
104 mÀ1 cmÀ1, yellow) reveals a larger absorption and a red
shift of 100 nm relative to that of TTP (lmax = 267 nm, e =
9.2 ” 103 mÀ1 cmÀ1, colorless). A smaller red shift was observed
for the 4-S-thymidine nucleotide (lmax = 335 nm, e = 2.0 ”
104 mÀ1 cmÀ1, colorless)[14,15] relative to the absorption of the
thymidine nucleotide. The color of the Se-modified nucleo-
tide is probably due to the ease of delocalization of the
selenium electrons on the nucleobase. Thus, less energy is
required for electron excitation, and the large red shift in the
UV spectrum results.
The Se-triphosphate is a good substrate for DNA
polymerases. It is recognized efficiently by the high-fidelity
Klenow fragment of E. coli DNA polymerase I (Figures 2 and
3).[16] We incorporated the Se-modified triphosphate into
DNA molecules on several different DNA templates and
found that the polymerized Se-DNA is yellow. By using a
short DNA template (T1) we incorporated SeTTP into a short
DNA primer (P1; Figure 2A) and confirmed the incorpora-
tion of the Se-modified nucleotide by MS analysis (Fig-
ure 2B). The mass difference between the DNA molecules
extended with a single SeTTP or TTP unit is 63 Dalton, which
indicates the incorporation of the Se-modified thymidine base
(mass difference for the replacement of O with Se: 79À16 =
63 Dalton).
Figure 3. Time course of the incorporation of TTP and SeTTP into DNA
with a DNA primer (P2, 5’-GCGTAATACGACTCACTATAG-3’) and the
Klenow enzyme on a DNA template (T2, 3’-CGCATTATGCTGAGTGA-
TATCCG-TTGGACTACTCCGGCTTTCCGGCTTTGCATGT-5’). a) Gel elec-
trophoresis; b) plot of the incorporation of TTP and SeTTP into DNA
with respect to time.
With a longer DNA template, we examined the incorpo-
ration of TTP and SeTTP into DNAwith time (Figure 3A) and
found that the SeTTP polymerization efficiency is similar to
that of native TTP (Figure 3B). Both the Klenow fragment
and the exo-Klenow fragment incorporate SeTTP with high
efficiency; no significant difference between these two
enzymes was observed. Thus, the high-fidelity enzymes[16]
are capable of recognizing the Se-modified nucleobase. This
result is consistent with the incorporation of S-modified
thymidine by a polymerase.[2b,c]
In summary, we have synthesized a visible (yellow)
nucleoside triphosphate, 4-Se-thymidine 5’-triphosphate, by
changing a single atom in the parent base. DNA polymerase
recognizes efficiently this Se-modified triphosphate and the
À
Se-mediated hydrogen bond (Se···H N). The incorporation
of SeTTP into DNA yields colored DNA and occurs with the
same level of efficiency as the incorporation of natural TTP.
The spectroscopic properties of visible SeTTP and its poly-
merization into DNA will shed new light on specific
recognition governed by the size and shape of bases, base
pairing, and stacking interactions, and on the efficiency and
fidelity of DNA replication. Moreover, the Se-DNA and its
visualization have great potential in the determination of
nucleic acid crystal structures via multiwavelength anomalous
dispersion (MAD) phasing,[3e,4,17] as well as for the nucleic
acid based detection of human diseases and pathogens.
Figure 2. Incorporation of SeTTP or TTP by the exo-Klenow enzyme on
a DNA template (T1, 3’-ATCGCCCAACGACCACCTTGG-5) with a
primer (P1, 5’-32P-TAGCGGGTTGCTGG-3’). a) All lanes contain P1 and
T1. Lane 1: no enzyme; lane 2: no TTP; lane 3: SeTTP and enzyme;
lane 4: TTP and enzyme. b) MS spectra of P1 (green; m/z calc. for P1:
4349.8 [MÀH]À; found: 4349), the T-extended DNA (O-15-mer, blue;
m/z 4662, 4815, 4966), and the SeT-extended DNA (Se-15-mer, red;
m/z 4726, 4876, 5028). T1: m/z 6345 (peak labeled in black). Mass
differences between the Se-15-mer and the O-15-mer: 4726À4662=64,
4876À4815=61, 5028À4966=62 (see the Supporting Information).
Received: November 12, 2007
Revised: December 6, 2007
Published online: January 18, 2008
Keywords: DNA modifications · DNA polymerization ·
.
nucleic acids · selenium · visualization
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