Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications

Article abstract of DOI:10.1021/ja408814g

We synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addition, the dTPT3 scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3^PA-dNaM pair, which is amplified only slightly less well. The identification of dTPT3 represents significant progress toward developing an unnatural base pair for the in vivo expansion of an organism's genetic alphabet and for a variety of in vitro biotechnology applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.

Full text of DOI:10.1021/ja408814g

Communication
pubs.acs.org/JACS
Natural-like Replication of an Unnatural Base Pair for the Expansion
of the Genetic Alphabet and Biotechnology Applications
Lingjun Li,† Melissa Degardin,† Thomas Lavergne,† Denis A. Malyshev,† Kirandeep Dhami,†
́
Phillip Ordoukhanian,‡ and Floyd E. Romesberg*,†
†Department of Chemistry and ‡Center for Protein and Nucleic Acid Research, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: We synthesized a panel of unnatural base
pairs whose pairing depends on hydrophobic and packing
forces and identify dTPT3-dNaM, which is PCR amplified
with a natural base pair-like efficiency and fidelity. In
addition, the dTPT3 scaffold is uniquely tolerant of
attaching a propargyl amine linker, resulting in the
dTPT3PA-dNaM pair, which is amplified only slightly
less well. The identification of dTPT3 represents
significant progress toward developing an unnatural base
pair for the in vivo expansion of an organism’s genetic
alphabet and for a variety of in vitro biotechnology
applications where it is used to site-specifically label
amplified DNA, and it also demonstrates for the first time
that hydrophobic and packing forces are sufficient to
mediate natural-like replication.
he four letter natural genetic alphabet is conserved
Tthroughout nature and is based on the complementary
shape and hydrogen-bonding (H-bonding) of the natural
nucleotides. Since its original proposal by Benner in 1990,1
the development of unnatural base pairs has become an active
area of research.2−7 Early efforts focused on novel pairs that
form via orthogonal H-bonding patterns, and progress along
this route continues.8 As an alternative approach, inspired in
part by Kool’s demonstration that H-bonds are not an absolute
requirement for replication,9 our group2,10−12 and the Hirao
group13−17 have demonstrated that hydrophobic and packing
forces are also sufficient to mediate unnatural base pair
replication. In particular, we have developed a class of unnatural
base pairs, exemplified by d5SICS-dNaM (Figure 1A), that
when incorporated into DNA are efficiently replicated without
sequence bias2,18 and efficiently transcribed into RNA.19,20
However, neither d5SICS-dNaM nor any of the other
unnatural base pairs reported to date are replicated with
natural base pair-like efficiencies or fidelities. In addition to
potentially compromising the potential uses of these unnatural
base pairs for both in vivo and in vitro applications, this raises
the fundamental question of whether hydrophobicity and
packing forces are truly sufficient to mediate replication with
natural-like efficiencies and fidelities.
Figure 1. (A) Previously identified unnatural base pairs. (B) Analogs
used in this study, with d5SICSPA shown for comparison.20
R=COCHCl2. Sugar and phosphate backbone are omitted for clarity.
for dMMO2, eventually yielding d5SICS. After progress stalled,
our efforts turned to the optimization of dMMO2 as a partner
for d5SICS, which eventually yielded dNaM.2,12,18 Although
the discovery of dNaM represented a significant improvement
in replication, continued optimization efforts again
stalled,12,18,21−23 implying that further optimization of dNaM
as a partner for d5SICS was unlikely. Thus, our focus turned to
the optimization of d5SICS as a partner for dNaM.
Structural studies in duplex DNA demonstrated that
d5SICS-dNaM forms via cross-strand intercalation.23,24 This
results in a structure that is more similar to a mispair than a
correct pair, making the efficient replication of the unnatural
base pair difficult to understand. However, recent studies have
shown not only that pairing of dNaM with d5SICSTP within a
polymerase active site induces the polymerase to undergo the
The d5SICS-dNaM pair was identified from the optimization
of dSICS-dMMO2 (Figure 1A), which was identified from a
screen of 3600 candidate unnatural base pairs.11 Early
optimization efforts focused on improving dSICS as a partner
Received: August 30, 2013
Published: October 23, 2013
© 2013 American Chemical Society
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Communication
Table 1. Kinetic and PCR Amplification Data
Presteady-state kinetics
OneTaq PCR (48 cycles)
Taq PCR (20 cycles)
a
b
dXTP
%inc
%ext
amplification ×1012
retention, %
96.3 1.7
fidelity, %
amplification ×103
retention, %
fidelity, %
5SICS
FPT1
57.0 0.2
7.2 0.2
28.7 0.5
65.7 0.5
72.3 0.5
66.3 0.5
68.3 0.4
7.0 0.2
15.1 1.1
32.0 1.5
8.8 0.2
34.5 0.5
49.8 1.3
33.8 0.2
31.5 0.7
5.5 0.1
9.4
9.7
99.91 0.04
98.89 0.09
98.8 0.3
7.7
10.9
6.7
86.7 1.0
42.0 1.1
98.90 0.01
93.73 0.18
91.81 0.01
99.46 0.01
99.66 0.13
99.9 0.2
62
60
2
7
TPT1
13.3
16.6
12.9
6.5
33.76 0.03
93.05 0.04
95.6 1.7
TPT2
96.8 0.9
>99
99.90 0.02
>99.98
9.9
TPT3
11.7
5.0
FTPT3
TPT3PA
5SICSPA
97.9 1.0
98.6 1.2
99.95 0.02
99.97 0.03
98
85
3
4
4.7
3.5
98.7 0.4
c
c
9.2
45
2
98.16 0.12
6.4
−
−
a
b
Incorporation assay conditions: 40 nM unnatural triphosphate, 2 μM dCTP, 10 s. Extension assay conditions: 10 μM unnatural triphosphate, 2
μM dCTP, 10 s. Unnatural base pair was lost during amplification.
c
open-to-closed structural transition characteristic of normal
synthesis, but also that within the closed complex, dNaM and
d5SICSTP pair in a Watson−Crick-like fashion.24 This
mutually induced fit mechanism is likely responsible for the
efficient incorporation of the unnatural triphosphate, but
structure−activity relationship (SAR) data suggested that,
after translocation within the polymerase binding site, in
preparation for incorporation of the next triphosphate, the
nascent unnatural base pair again returns to an intercalated
state, making deintercalation a requirement for continued DNA
synthesis.12 Thus, we reasoned that further optimization might
be possible with d5SICS analogs that conform to the SAR rules
governing efficient base pair synthesis (via triphosphate
incorporation) and extension (via incorporation of the next
triphosphate), but that are less prone to intercalate. As SAR
data have clearly demonstrated that the thio N-glycoside of
d5SICS is required for both synthesis and extension, we
reasoned that modifying and/or reducing the aromatic surface
of the second ring might represent a route to favor
deintercalation.
To explore the reduction of the aromatic surface area of the
d5SICS scaffold, as well as to systematically vary nucleobase
dipole moment and polarizability, we synthesized the furano
and thieno substituted pyridine-2(1H)-thione heterocycles
dFPT1, dTPT1, and dTPT2 (Figure 1B, Supporting
Information (SI)). Briefly, the nucleobase analogs were
synthesized via an intermolecular Curtius rearrangement and
then coupled to (2R,5R)-5-chloro-2-(((4-methylbenzoyl)oxy)-
methyl) tetrahydrofuran-3-yl 4-methylbenzoate, resulting in a
mixture of anomeric nucleosides, with the pure β-anomer
obtained by column chromatography. After sulfonylation and
toluyl deprotection, the free nucleosides were converted to
triphosphates under Ludwig conditions and purified by anion
exchange chromatography and reversed-phase HPLC.
In previous work, we employed steady-state kinetics to
analyze both unnatural base pair synthesis and extension.
However, the chemical steps underlying d5SICS-dNaM
synthesis and extension are sufficiently efficient that, under
steady-state conditions, product formation is limited by
dissociation,25 rendering the steady-state kinetics data less
helpful for the optimization of processive replication. Thus, we
analyzed the new derivatives using a presteady-state assay.22
The assay is based on determining, under a fixed set of
conditions, the amount of a 23-mer primer that is extended by
addition of the unnatural triphosphate opposite its cognate
nucleotide in a 45-mer template by the Klenow fragment of E.
coli DNA polymerase I (Kf). The efficiency of unnatural base
pair synthesis is characterized by measuring the percent
incorporation (%inc) at a given concentration of the unnatural
and next correct triphosphate (dCTP), determined from the
ratio [24-mer + 25-mer]/[23-mer + 24-mer + 25-mer]. The
efficiency of extension is characterized by measuring the
percent extension (%ext) at a given concentration of the next
correct triphosphate (dCTP) and saturating concentrations of
unnatural triphosphate, determined from the ratio [25-mer]/
[24-mer + 25-mer]. Under the conditions selected, d5SICSTP
is incorporated opposite dNaM with a %inc of 57, and the
resulting d5SICS-dNaM pair is extended with a %ext of only
15. The derivatives showed a wide range of behaviors (Table
1). dFPT1TP is incorporated less efficiently, but dFPT1-dNaM
is then extended more efficiently, while the opposite is true for
dTPT1TP. However, dTPT2TP is incorporated more
efficiently than d5SICSTP, and the subsequently formed
dTPT2-dNaM pair is also extended more efficiently than
d5SICS-dNaM.
Based on these data we further explored the dTPT2 scaffold
with dTPT3 (Figure 1B). With this analog, removal of the
methyl group was expected to further modify the tendency of
the nucleobase to cross-strand intercalate, and it was
synthesized by adaptation of the dTPT2 synthesis (SI).
Interestingly, both dTPT3TP incorporation opposite dNaM
and extension of the resulting unnatural pair are more efficient
than with dTPT2TP (Table 1).
To more fully evaluate replication, the previously reported
template D6,2,26 containing either d5SICS or a d5SICS analog
paired opposite dNaM, was amplified by PCR (in this template,
the unnatural base pair is flanked on each side by three
randomized natural nucleotides, SI). Two sets of PCR reactions
were explored (Table 1), one employing 48 cycles with
OneTaq polymerase, a commercially available mixture of
exonuclease-negative Taq polymerase and exonuclease-positive
DeepVent polymerases, and one employing 20 cycles of
amplification with exonuclease-negative Taq alone, to explore
the contribution of proofreading. Efficiency was determined by
monitoring the amplification level, and fidelity (defined as
unnatural base pair retention per doubling) was determined
from the percentage of the amplified DNA that retained the
unnatural base pair, which was determined from the relative
peak intensities of a sequencing chromatogram (SI and
Malyshev et al.26). For comparison, with OneTaq natural
DNA is amplified 5.5 × 1013-fold and DNA containing d5SICS-
dNaM is amplified 9.4 × 1012-fold, and 96.3% of the DNA
retained the unnatural pair, corresponding to a fidelity of
99.91%. Under these conditions DNA containing dFPT1-
dNaM or dTPT1-dNaM is amplified ∼1 × 1013-fold, but with
fidelity that is slightly reduced relative to d5SICS-dNaM
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Figure 2. Determination of postamplification DNA labeling efficiency via streptavidin (SA) gel shift. (A) Labeling strategy (illustrated for dTPT3PA
-
dNaM; B = biotin). (B) Labeling with unnatural base pair centrally located in 134-mer duplex. The faster and slower migrating bands correspond to
dsDNA, and the 1:1 complex between dsDNA and streptavidin, respectively. (C) Labeling with unnatural base pair positioned at first, ninth, or
eleventh positions, corresponding to duplexes 1−3, respectively.
(∼98.8%). However, amplification of DNA containing dTPT2-
dNaM proceeded more efficiently than d5SICS-dNaM, and
with an indistinguishable fidelity. Amplification of DNA
containing dTPT3-dNaM again proceeded more efficiently
than d5SICS-dNaM, and remarkably, with no mispaired
sequences detected. With a lower limit of detection estimated
to be one mispaired sequence in 102 (corresponding to 99%
retention), this places a lower limit of 99.98% on the fidelity of
dTPT3-dNaM replication.
Due to the absence of proofreading, amplification with only
Taq polymerase is more likely to differentiate the unnatural
base pairs. Under the conditions employed with only Taq,
natural DNA is amplified 2.8 × 104-fold and DNA containing
d5SICS-dNaM is amplified 7.7 × 103-fold and with a fidelity of
98.90%. DNA containing dFPT1-dNaM or dTPT1-dNaM is
amplified with significantly lower fidelity. However, DNA
containing dTPT2-dNaM, and especially dTPT3-dNaM, is
amplified with greater efficiency and fidelity. In fact, dTPT3-
dNaM is amplified with a fidelity that is only slightly less than
that observed with OneTaq. Clearly, based on the presteady-
state kinetics and both PCR conditions, dTPT2 and especially
dTPT3 are significantly better optimized as partners for dNaM
than d5SICS or any of the other analogs examined.
In principle, an unnatural base pair should allow for the site-
specific inclusion of different functionalities into DNA, either
pre- or postamplification,14,20,27 for biophysical, SELEX
(Systematic Evolution of Ligands by Exponential Enrich-
ment)28,29 or nanomaterial applications.30 To explore the
potential of the dTPT3 scaffold for such applications, we
synthesized dFTPT3TP and dTPT3PATP (Figure 1B), with the
former installing an NMR active nuclei for biophysical
characterization31,32 and the latter bearing a linker for the
site-specific modification of the amplified DNA. Both analogs
were obtained from the toluyl-protected nucleoside of dTPT3
(SI). Briefly, dFTPT3 was obtained via fluorination, sulfona-
tion, benzoyl deprotection, and phosphorylation, to yield the
corresponding triphosphate. dTPT3PATP was obtained via
iodination, sulfonylation, coupling with the dichloro acetyl
propargylamine, deprotection, and phosphorylation.
Both fluoro and propargyl amine substituents have similar
effects on the presteady-state kinetics, slightly reducing the
%inc opposite dNaM, and more substantially reducing the %ext
(Table 1). However, %inc and %ext for both analogs remain
significantly greater than those for d5SICS. Interestingly, DNA
containing either dFTPT3-dNaM or dTPT3PA-dNaM is PCR
amplified with nearly the same fidelity as dTPT3-dNaM, and
remarkably, both are amplified as well or better than DNA
containing d5SICS-dNaM (Table 1). In fact, with Taq alone,
dFTP3-dNaM is better replicated than any previously reported
unnatural base pair. The ability of the dTPT3 scaffold to
support the propargyl amine linker is particularly remarkable, as
dTPT3PA-dNaM is replicated significantly better than
d5SICSPA-dNaM (Figure 1B), which previously was the most
efficiently amplified linker-bearing unnatural base pair.20 With
OneTaq, the fidelity is 99.97% versus 98.16% per doubling for
dTPT3PA-dNaM and d5SICSPA-dNaM, respectively. Dramati-
cally, with Taq alone, d5SICSPA-dNaM is lost during
amplification while dTPT3PA-dNaM is amplified with a fidelity
of 98.7%.
Finally, to explore the use of dTPT3PA for the site-specific
labeling of DNA, we examined the amplification, deprotection,
and labeling of a 134-mer DNA containing a centrally
positioned dTPT3PA-dNaM or d5SICSPA-dNaM. Each tem-
plate was amplified ∼500-fold, deprotected, coupled to NHS-
PEG4-biotin, and analyzed by gel shift with streptavidin (Figure
2A and B). With d5SICSPA-dNaM, 72% of the DNA was
shifted, while, for dTPT3PA-dNaM, 80% of the DNA was
shifted. To explore site-specific labeling at potentially more
challenging positions, dTPT3PA-dNaM or d5SICSPA-dNaM
was incorporated into a 60-mer DNA at the first, ninth, and
eleventh position, resulting in duplexes 1−3, respectively
(Figure 2C). With d5SICSPA-dNaM, 6%, 56%, and 84% of
the amplified DNA was shifted, while, with dTPT3PA-dNaM,
72%, 81%, and 94% was shifted. Clearly, the more efficient
amplification of dTPT3PA-dNaM, especially when it is located
near the end of a template, results in a significantly greater
efficiency of site-specific labeling. This should facilitate many
applications, including those requiring that both ends of the
same DNA strand are modified, which is otherwise challenging.
In summary, we found that contraction of the distal phenyl
ring of d5SICS to a methyl thieno ring significantly increases
replication and that subsequent removal of the methyl group
increases it still further. These effects are clearly dependent on
the detailed structure of the nucleobase. Moreover, it is
noteworthy that dTPT3 represents a rare example among
predominantly hydrophobic unnatural base pairs where a
reduction in hydrophobicity facilitates replication. A simple
explanation for this SAR is that when appropriately positioned,
the sulfur atom increases intrastrand packing interactions, while
the methyl group favors interstrand packing interactions. Thus,
both addition of the sulfur and removal of the methyl group are
expected to stabilize the transition state for incorporation and
also favor deintercalation of the nascent unnatural base pair,
which our model predicts would facilitate extension.12 The
robust accommodation of substituents within the dTPT3
scaffold is also consistent with this model, since only when the
pair adopts a Watson−Crick-like structure is a substituent at
828
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis, characterization, presteady-state kinetic data, PCR
assay, and analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
floyd@scripps.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH (GM 060005).
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