Amplification of 4′-thioDNA in the presence of 4′-thio-dTTP and 4′-thio-dCTP, and 4′-thioDNA-directed transcription in vitro and in mammalian cells

Article abstract of DOI:10.1021/ja075953c

Herein we describe the synthesis of 4′-thio-dTTP (1) and 4′-thio-dCTP (2) and their susceptibility for PCR amplification. Double stranded 4′-thioDNAs were amplified sufficiently by KOD dash DNA polymerase under appropriate conditions. Through this PCR stu

Full text of DOI:10.1021/ja075953c

Published on Web 11/23/2007
Amplification of 4′-ThioDNA in the Presence of 4′-Thio-dTTP and
4′-Thio-dCTP, and 4′-ThioDNA-Directed Transcription in Vitro and in
Mammalian Cells
Naonori Inoue,† Aki Shionoya,§ Noriaki Minakawa,*,† Akiko Kawakami,§ Naoki Ogawa,§ and
Akira Matsuda*,†
Graduate School of Pharmaceutical Sciences, Hokkaido UniVersity, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812,
Japan, and GeneticLab Co., Ltd, Kita-27, Nishi-6, Kita-ku, Sapporo 001-0027, Japan
Received August 8, 2007; E-mail: noriaki@pharm.hokudai.ac.jp; matuda@pharm.hokudai.ac.jp
A number of modified oligonucleotides (ONs), prepared by a
general chemical approach using the corresponding phosphoramidite
units, have been synthesized in order to evaluate their functions.
An alternative enzymatic method using the corresponding nucleo-
side triphosphates could also be used. Since this approach affords
long chain sequences from readily available natural DNA templates,
if successful, it would be useful in a large number of biotechnolo-
Figure 1. Structures of dSTTP (1) and dSCTP (2).
gies.1 Therefore, intense efforts are currently underway toward an
efficient enzymatic synthesis of modified ONs.2 However, this
approach may have limited use because of the low-substrate
specificity of the modified nucleoside triphosphates to DNA/RNA
polymerases.
Our group synthesized 4′-thioDNA (SDNA), a modified DNA
analogue which showed nuclease resistance and hybridization
ability.3 Prompted by these and other favorable properties, we
decided to explore the enzymatic synthesis of SDNAs and their
function. We now wish to report the amplification of double
stranded SDNAs (ds-SDNAs)4 by PCR in the presence of 4′-thio-
Figure 2. Yield of ds-SDNA amplified by PCR using Taq, Therminator,
and KOD dash DNA polymerases in the presence of dSTTP (1) and/or
dTTP (dSTTP; 1) and 4′-thio-dCTP (dSCTP; 2) (Figure 1). The
dSCTP (2). The reaction mixture contains dTTP, dCTP, dATP, and dGTP
ability of the resulting ds-SDNAs as templates to afford natural
(lane 1); 1, dCTP, dATP, and dGTP (lanes 2 and 5); dTTP, 2, dATP, and
RNA in vitro and in mammalian cells will also be described.
The desired triphosphates 1 and 2 were prepared from the
appropriately protected 2′-deoxy-4′-thio derivatives5 in a manner
similar to that described previously6 (Supporting Information,
Scheme S1). Prior to PCR amplification, primer extension reactions
of 1 and 2 were examined using various thermophilic enzymes
((family A) Taq, Tth, and Tfl DNA polymerases; (family B) Vent,
Deep Vent, Pfu, Tli, Therminator, and KOD dash DNA poly-
merases). Among the enzymes examined, the family B enzymes
such as Therminator and KOD dash DNA polymerases were found
to be the most favorable for incorporation of 1 and 2 (data not
shown).2a With these results in hand, we examined the amplification
of ds-SDNAs in the presence of 1 and/or 2 using Therminator and
KOD dash DNA polymerases in addition to the Taq DNA
polymerase for comparison. The sequences of the 87mer template
and the appropriate primers, one of which contained the promoter
sequence of the T7 RNA polymerase for a subsequent transcription
experiment, to afford the 104mer amplified ds-DNA are presented
in the Supporting Information (Figure S1).4 The resulting ds-SDNAs
were analyzed quantitatively, and the results are summarized in
Figure 2 (and Table S1). When amplification was examined under
the usual conditions (condition I; 72 °C for 15 s), neither Taq DNA
polymerase nor Therminator and KOD dash DNA polymerases
afforded the amplified product in sufficient yields compared with
experiments carried out in the presence of the four natural dNTPs,
dGTP (lanes 3 and 6); and 1, 2, dATP, and dGTP (lanes 4 and 7). The
reaction conditions (conditions I and II) are described in Supporting
Information.
except for KOD dash in the presence of 2. To improve efficiency,
reaction conditions, such as temperature and reaction time of each
PCR cycle, were examined. As a result, increasing the reaction time
of each cycle from 15 s to 30 min (condition II; 65 °C for 30 min),
produced a drastic improvement in amplification even in the
presence of both 1 and 2, especially when KOD dash DNA
polymerase was used. Under these modified conditions, only the
amplified ds-SDNAs,4 whose lengths were confirmed by PAGE
analysis (Figure S2), were obtained in the presence of 1 and/or 2
even under long reaction times such as these. Through this PCR
study, it was shown that not only DNA-directed SDNA synthesis
but also SDNA-directed SDNA synthesis occurred in the presence
of 1 and/or 2.
In subsequent investigations, we tested whether the resulting ds-
SDNA acted as a template for transcription by RNA polymerases.
In vitro transcription by the T7 RNA polymerase in the presence
of the four natural NTPs using the aforementioned PCR products
as templates was first examined to afford an 87mer RNA sequence
(Figure S1). As can be seen in Figure 3 (and Figure S3), all ds-
SDNAs4 as templates afforded the same length of RNA, and the
transcription efficiency was estimated in 38-49% yield relative to
in the case of natural DNA template.
These results prompted us to further investigate whether the
transcription occurred in cells. Accordingly, we constructed a certain
† Hokkaido University.
§ GeneticLab Co.
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10.1021/ja075953c CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
come from nuclease resistance of ds-SDNA.3 With more research,
ds-SDNA could be developed as a new DNA device for gene-
silencing.
In conclusion, we prepared 4′-thio-dTTP and 4′-thio-dCTP and
investigated their susceptibility for PCR amplification. Ds-SDNAs
were amplified sufficiently by KOD dash DNA polymerase under
appropriate conditions. The resulting 4′-thioDNAs acted as tem-
plates for transcription not only in vitro but also in mammalian
cells to afford RNA which showed gene-silencing effects via RNAi
machinery. Unlike nucleoside triphosphates modified on their
nucleobase and phosphorus positions,8,9 no example of PCR
amplification using sugar-modified nucleoside triphosphates is
known. While pioneering investigations of PCR amplification using
RS-dNTPs and in vitro transcription has been carried out using the
resulting modified DNA,10 to the best of our knowledge, the
potential of ds-SDNA for transcription in cells as reported in this
study is the first reported application with modified nucleic acids.
Thus, these results suggested the importance of SDNA when
implementing an enzymatic approach to produce not only aptamers
by SELEX but also new DNA devices for gene-silencing via RNAi
machinery.
Figure 3. Ultraviolet image from ethidium bromide-stained 2.5% agarose
gel of the transcriptional products by T7 RNA polymerase (Full image of
the gel was presented in the Supporting Information). The reaction mixture
contains DNA104 as a template and NTPs (lane 1); SDNA104+1 as a
template and NTPs (lane 2); SDNA104+2 as a template and NTPs (lane
3); and SDNA104+1,2 as a template and NTPs (lane 4). The transcriptional
products were quantified by RiboGreen as 0.47 µg/mL (lane 1, 100%), 0.23
µg/mL (lane 2, 49%), 0.21 µg/mL (lane 3, 45%), and 0.18 µg/mL (lane 4,
38%).
Figure 4. Gene silencing of the expression of pGL3 luciferase gene using
scramble ds-DNA (lane 1), natural ds-DNA (lane 2), SDNA+1 (lane 3),
SDNA+2 (lane 4), and SDNA+1,2 (lane 5). Data are averages of three
independent experiments.
Acknowledgment. This work was supported in part by Grant-
in-Aid from the Japan Society for Promotion of Science.
Supporting Information Available: Synthesis of 1 and 2, PCR
amplification, in vitro transcription, and transcription in NIH/3T3 cells
resulting gene-silencing. This material is available free of charge via
the Internet at http://pubs.acs.org.
sequence of ds-DNA (or ds-SDNAs) by PCR, which was expected
to act as templates for transcription in cells by RNA polymerase
III (pol III), with the resulting short-hairpin RNA (shRNA) showing
a gene-silencing effect via RNAi machinery.7
References
Thus, only specific parts of the shRNA expression vector
(pSIREN-Shuttle-Luc) consisting of a U6 promoter and a gene of
interest (pGL3 luciferase) were amplified by PCR in the presence
of 1 and/or 2 (approximately 300 bp in length). The efficiency of
amplification of the ds-SDNAs4 relative to the natural ds-DNA was
as follows: SDNA+1, 51%; SDNA+2, 99%; SDNA+1,2, 11%.
The resulting ds-SDNAs were then used for gene-silencing experi-
ments. Cotransfection of individually amplified ds-SDNA (or ds-
DNA) and reporter plasmids (pGL3 control vector) into NIH/3T3
cells was carried out, and the potency of gene-silencing, RNAi
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Supporting Information). As shown in Figure 4, the natural ds-
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