Study on suitability of KOD DNA polymerase for enzymatic production of artificial nucleic acids using base/sugar modified nucleoside triphosphates.

Abstract

Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2'-ONHCH₂CH₂-4' linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

Full text

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Molecules 2010, 15, 8229-8240; doi:10.3390/molecules15118229


molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Study on Suitability of KOD DNA Polymerase for Enzymatic
Production of Artificial Nucleic Acids Using Base/Sugar
Modified Nucleoside Triphosphates
Masayasu Kuwahara 1,*, Yuuki Takano 1, Yuuya Kasahara 1, Hiroki Nara 1, Hiroaki Ozaki 1,
Hiroaki Sawai 1, Akio Sugiyama 2 and Satoshi Obika 3

1 Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515,
Japan
2 Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047,
Japan
3 Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
* Author to whom correspondence should be addressed; E-Mail: kuwahara@chem-bio.gunma-u.ac.jp;
Tel.: +81-277-30-1222; Fax: +81-277-30-1222.
Received: 30 September 2010; in revised form: 6 November 2010 / Accepted: 10 November 2010 /
Published: 12 November 2010

Abstract: Recently, KOD and its related DNA polymerases have been used for preparing
various modified nucleic acids, including not only base-modified nucleic acids, but also
sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be
promising candidates for nucleic acid drugs. However, thus far, reasons for the
effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated.
Therefore, using mutated KOD DNA polymerases, we studied here their catalytic
properties upon enzymatic incorporation of nucleotide analogues with base/sugar
modifications. Experimental data indicate that their characteristic kinetic properties
enabled incorporation of various modified nucleotides. Among those KOD mutants, one
achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-
4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for
modified nucleoside triphosphates were shown, and the effectiveness of genetic
engineering in improvement of the enzyme for modified nucleotide polymerization has
been demonstrated.
OPEN ACCESS

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Molecules 2010, 15
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Keywords: KOD DNA polymerase; artificial nucleic acids; modified nucleoside
triphosphates

1. Introduction
To diversify the ability and enhance the nuclease resistance of functional nucleic acids like
ribozymes and aptamers [1–4], enzymatic production of various modified nucleic acids has been
investigated [5–15]. This is because those functional nucleic acids can be created by a random
screening method, which involves enzymatic amplification of selected nucleic acids as a key step. We
first demonstrated that KOD Dash DNA polymerase is very suitable for amplifying modified DNA by
polymerase chain reaction (PCR) using the C5-substituted thymidine analogue [16]. Until now, many
types of DNA polymerases were evaluated, and some of them, e.g. Vent(exo-), Pwo and Phusion High-
Fidelity, were found to be useful for enzymatic production of artificial nucleic acids. However, a series
of experimental results from our laboratories indicated that KOD Dash DNA polymerase was more
tolerant than any of the other aforementioned polymerases, not only towards base-modification, but
also sugar-modification, and thus would be excellent for this purpose [17–20]. This is further
supported by recent reports [21, 22] regarding enzymatic production of bridged/locked nucleic acid
(BNA [23, 24]/LNA [25]) using KOD DNA polymerase. Furthermore, KOD DNA polymerase was
often reported as the only one working for long PCR with modified nucleoside triphosphates [26, 27].
KOD Dash DNA polymerase is a mixture of wild-type KOD DNA polymerase and KOD(exo-)
DNA polymerase having no or less 3′,5′ exonuclease activity. The wild-type KOD DNA polymerase
belongs to the evolutional family B, and was originally isolated from the hyperthermophilic archaeon,
Pyrococcus kodakaraensis [28]. Here, we have prepared eight KOD mutants (KOD1–8) [29, 30],
among which four mutants (KOD1–3, 8) have no or less 3′,5′ exonuclease activity, by genetically
engineering KOD DNA polymerase, then assessed and compared the catalytic properties of those
mutants, using thymidine analogues 1–3 (Figure 1) as substrates. Wild-type KOD DNA polymerase
(Toyobo), KOD Dash DNA polymerase (Toyobo), and Vent(exo-) DNA polymerase (New England
Biolabs) were also used as reference materials; Vent(exo-) is a genetically engineered form of the
native DNA polymerase, which also belongs to family B, from Thermococcus litoralis [31, 32].
Figure 1. Chemical structures of the thymidine 5′-triphosphate analogues used in this experiment.

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2. Results and Discussion
First, we performed modified nucleotide standing-start experiments under conditions that achieve a
single completed hit using template DNA T1 (5′- CCG AAT AAA GGT ATG GCC TGT TCG CTA
ATC C -3′) and 5′-(6-FAM)-labelled primer P1 (5′- GGA TTA GCG AAC AGG CCA TAC CTT -3′).
Four KOD mutants (KOD1–3, 8) and Vent(exo-) DNA polymerase were used as the enzyme, and
thymidine analogues 1 and 2, thymidine-5′-triphosphate (TTP) and 2′-deoxycytidine-5′-triphospate
(dCTP), were used as the substrates. The reaction was performed at 40°C because the reactions
proceeded too fast to be monitored at the optimal temperature of the enzyme (~75°C) [33]. All
reactions were performed under the same enzyme concentration of 0.0125 U/μL. The apparent values
for Km and Vmax were obtained from the initial velocities of the primer extension reactions, and then the
apparent insertion efficiency (Vmax/Km) of each substrate triphosphate usage was calculated. The
relative apparent insertion efficiency (accuracy) was defined as the ratio of modified analogue
insertion to TTP correct insertion efficiencies (Vmax/Km)analogue/(Vmax/Km)TTP, or that of dCTP
misinsertion to TTP correct insertion efficiencies (Vmax/Km)dCTP/(Vmax/Km)TTP as shown in Table 1.
Table 1. Modified/natural nucleotide incorporation in primer extension reaction using
KOD mutants and Vent(exo-) DNA polymerase. a
DNA
polymerase
KOD1



KOD2



KOD3



KOD8



Vent(exo-)



a Experimental conditions are described in Experimental Section. b The initial rate relative apparent
insertion efficiency (accuracy); (Vmax/Km)analogue/(Vmax/Km)TTP and (Vmax/Km)dCTP/(Vmax/Km)TTP.

dNTP
Vmax
(min–1)
1.8
11
0.10
0.074
2.1
4.0
0.32
0.019
2.3
4.4
0.27
0.033
9.5
53
2.4
0.11
0.34
0.29
0.049
0.14
Km
(mM)
0.021
0.14
0.26
0.66
0.035
0.083
0.099
0.11
0.047
0.081
0.099
0.70
0.063
0.31
0.38
1.0
0.0030
0.0027
0.0044
1.7
Insertion efficiency
Vmax/Km
88
81
3.8
0.11
60
48
3.3
0.17
49
55
2.7
0.048
150
170
6.3
0.11
110
110
11
0.083
Accuracyb
T
1
2
C
T
1
2
C
T
1
2
C
T
1
2
C
T
1
2
C
1
0.92
0.043
0.0013
1
0.80
0.055
0.0029
1
1.1
0.055
0.00097
1
1.1
0.041
0.00073
1
0.95
0.099
0.00073

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In incorporation of thymidine analogues 1 and 2, all KOD mutants used showed apparent Km values
larger than that for natural TTP; those values were about 1.7- to 6.4-fold in the cases of the base-
modified analogue 1, and were about 2.1- to 13-fold in the cases of the base- and sugar-modified
analogue 2. On the other hand, Vent(exo-) DNA polymerase showed Km values for those two analogues
that were almost same as that for TTP. In misincorporation of dCTP, apparent Km values of KOD
mutants were only 3.2- to 32-fold larger than that for TTP as a correct incorporation substrate, while
that of Vent(exo-) was about 580-fold larger than that for TTP. However, relative insertion efficiency
(accuracy), in other words, substrate specificity of polymerase of when insertion of thymidine
nucleotide is the correct incorporation, were almost the same between KOD mutants and Vent(exo-)
DNA polymerase. This is because KOD mutants provide larger Km and Vmax in the cases of analogue
incorporation, and smaller Km and Vmax in the cases of the misincorporation, compared with Vent(exo-)
DNA polymerase. As a result, only slight differences in the ratios of Km to Vmax were seen between
KOD mutants and Vent(exo-) DNA polymerase. This catalytic property of KOD mutants would be
favourable for enzymatic production of artificial nucleic acids, that is, large Vmax values of KOD
mutants in analogue incorporation per misincorporation indicate that the reaction of analogue
incorporation could progress much faster than that of dCTP misincorporation under sufficiently high
substrate concentrations.
We then investigated successive incorporations of modified nucleotide by primer extension
reactions using bridged nucleotide 3, because bridged/locked nucleic acids (BNA/LNAs) would be
promising candidates for nucleic acid drugs [34, 35]. We have previously reported [36] that
incorporation of this type of bridged nucleotide (2′,4′-BNANC) [37] is more difficult than that of the
prototype 2′-O,4′-C-methylene bridged/locked nucleotide (BNA/LNA), which can be incorporated
successively using wild-type KOD DNA polymerase [23]. However, 2′,4′-BNANC–type nucleotides
possess excellent nuclease resistance much superior to the prototype [38, 39]; therefore, its
applications in random screening of nucleic acid enzymes and aptamers, which would require efficient
enzymatic incorporation of modified nucleotides, are highly anticipated. In this experiment, template
DNA T2 (5′- CCG AAA AAA GGT ATG GCC TGT TCG CTA ATC C -3′), 5′-(6-FAM)-labeled
primer P2 (5′- GGA TTA GCG AAC AGG CCA TAC CTT T -3′) and eight KOD mutants (KOD1–8)
and Vent(exo-) DNA polymerase were used.
As shown in Figure 2, wild-type KOD DNA polymerase, KOD4, KOD5 and KOD7 exhibited the
strong 3′,5′ exonuclease activity [40], and did not provide any elongated products, while the other
polymerases provided some elongated products. Among these, KOD8 mainly provided a longer
elongated product; it would be four-successive incorporations although three-successive incorporated
products must be provided as the full-length product under the condition using those materials. This
may have occurred because of DNA template slippage caused by conformation of DNA/BNA helix
different from the natural DNA/DNA helix between primer and template strands [41]. Note that the
efficient multiple-successive incorporation of bridged analogue 3 under severe conditions such as
without any additives, e.g. manganese chloride and betaine [42, 43], could be made by one of KOD
mutants assessed, KOD8.

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Figure 2. Successive incorporation of 2′,4′-bridged nucleotides using analogue 3 with
various DNA polymerases; KOD Dash (lane 2), wild type KOD (lane 3), KOD1 (lane 4),
KOD2 (lane 5), KOD3 (lane 6), KOD4 (lane 7), KOD5 (lane 8), KOD6 (lane 9), KOD7
(lane 10), KOD8 (lane 11), and Vent(exo-) (lane 12). Primer extension reactions were
performed for 1 h at 74°C under enzyme concentration of 0.4 U/μL. Primer P2 only
migrated in lane 1.












3. Experimental
3.1. Synthesis of modified nucleoside triphosphate analogs and their intermediates
The modified nucleoside triphosphates 1 and 3 were synthesized according to our previous reports
[16, 36]. The synthetic routes of the modified nucleoside triphosphate 2 and its intermediates are
shown in Scheme 1. NMR Spectra were recorded on a JEOL JNM-AL300 FT-NMR spectrometer.
Mass spectral analysis for nucleoside analogues was accomplished on an MDS-Sciex API-100
spectrometer (Applied Biosystems) under atmospheric pressure ionization conditions. Thin-layer
chromatography was performed using silica gel 60 F254 plates (Merck). Reversed-phase high-
performance liquid chromatography (HPLC) was performed using a JASCO Gulliver system with UV
detection at 260 nm and a packed Wakosil 5C18 (φ 4.6×250 mm; Wako) or TSKgel ODS-80Ts
(φ 20×250 mm; Tosoh) column. Silica gel 60 (Kanto Chemical; 40–50 μm) was used for normal-
phase column chromatography, and Wakosil 40C18 (Wako) was used for reversed-phase column
chromatography. Reversed-phase medium-pressure liquid chromatography (MPLC) was performed
using an YFLC-Wprep system (Yamazen) with a glass column (φ 33×250 mm) filled with Wakosil
40C18 (Wako). Ion exchange column chromatography was performed using an ECONO system (Bio-
Rad) with a glass column (φ25×500 mm) filled with diethylaminoethyl (DEAE) A-25-Sephadex
(Amersham Biosciences).