Efforts towards expansion of the genetic alphabet: Pyridone and methyl pyridone nucleobases

Article abstract of DOI:10.1002/anie.200601272

(Figure Presented) Getting on with it: A common problem with hydrophobic non-natural base pairs - the inefficient extension following such a base pair - is reduced by using the methylpyridone scaffold, which simultaneously limits another frequent problem

Full text of DOI:10.1002/anie.200601272

Communications
Non-natural Base Pairs
DOI: 10.1002/anie.200601272
Efforts towards Expansion of the Genetic
Alphabet: Pyridone and Methyl Pyridone
Nucleobases**
Aaron M. Leconte, Shigeo Matsuda, Gil Tae Hwang,
and Floyd E. Romesberg*
A non-natural base pair, which is stable in duplex DNA and
enzymatically synthesized with high efficiency and selectivity,
would greatly expand the utility of nucleic acids, both in terms
of their genetic-coding capacity and chemical functionality.
Unlike the natural base pairs, a third base pair does not
[
1]
[2]
[3]
necessarily require H-bonding, and we, and others, have
demonstrated that hydrophobic and van der Waals forces can
mediate the selective interbase interactions required for base-
pair stability and synthesis.
Our early efforts focused on nucleotides bearing nucleo-
base analogues with large extended aromatic surfaces. A
variety of self-pairs (formed between the same analogue) and
heteropairs (formed between different analogues) were
identified. These pairs were both stable in duplex DNA and
efficiently synthesized by the exonuclease deficient Klenow
[
2a,4]
fragment of DNA polymerase I (Kf).
However, replica-
tion was consistently limited by poor extension (i.e. continued
primer elongation after synthesis of the non-natural pair),
which we reasoned could be the result of interbase interca-
lation of the large aromatic scaffolds that distort the newly
formed primer terminus and prevent its efficient extension.
Our second-generation non-natural base pairs were formed
between nucleotides that bear small, aromatic nucleobase
analogues that were designed to be too small to intercalate
and therefore more likely to interact edge on. Although
several base pairs have been found that are more efficiently
[
2b,5]
extended by Kf, the rates remain insufficient.
Further-
[*] A. M. Leconte, S. Matsuda, G. T. Hwang, Prof. Dr. F. E. Romesberg
Department of Chemistry
The Scripps Research Institute
La Jolla, CA 92037 (USA)
Fax: (+1)858-784-7472
E-mail: floyd@scripps.edu
[**] Acknowledgement for financial support is given to the National
Institutes of Health (GM60005 to F.E.R.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
[
a]
more, many of the smaller analogues bearing small hydro-
phobic nucleobases are also limited by facile and stable
Table 1: T
m
values of DNA duplexes.
’-dGCGTACXCATGCG
3’-dCGCATGYGTACGC
5
[
2b,11]
mispairing with dA.
Thus, although small aromatic
nucleobase analogues are promising, further optimization is
required.
X
Y
T
m
[8C]
X
Y
T [8C]
m
3
3
MP
MP
3MP
A
C
G
T
4MP
A
C
G
T
T
53.4
49.4
46.4
51.4
49.4
50.4
48.4
44.4
52.4
47.2
59.2
5MP
5MP
5MP
5MP
5MP
PYR
PYR
4MP
PYR
PYR
A
5MP
A
C
G
T
PYR
A
C
G
T
49.3
48.3
43.3
52.3
47.3
50.2
49.2
43.2
52.2
47.2
55.4
One factor that may contribute to the reduced Kf-
mediated extension of the small aromatic pairs is their
inability to engage the polymerase at a primer terminus. The
natural base pairs have a similar geometric shape with a
hydrogen bond acceptor located in an analogous position in
the minor groove, N3 in purines or O2 in pyrimidines. When
at the primer terminus, these H-bond acceptors have been
shown to be important for polymerase-mediated exten-
3MP
3MP
3
4
4
4
4
MP
MP
MP
MP
MP
4MP
A
[
6,7]
[10]
[10]
sion.
Herein, we report the synthesis and characterization
G
of nucleotides bearing pyridone-nucleobase analogues that
are expected to position their carbonyl group into the minor
groove (Figure 1a).
[
a] For experimental details, see the Supporting Information.
of the mispairs with dG. The relative stability of the mispairs
with dG is likely due to the increased aromatic surface area of
guanine, as well as its exocyclic minor-groove amine, which
may H-bond with the pyridone-nucleobase analogues. In
contrast, the fully carbocyclic analogues form their most
[
9]
stable mispairs with dA, likely due to the increased hydro-
phobicity of adenine relative to the other natural nucleobases.
Relative to substitution at the 4- or 5-position, substitution at
the 3-position has a more variable effect on mispair stability:
it has little effect on the mispair with dA, stabilizes the
mispairs with dC and dT, and destabilizes the mispair with dG.
The stability of the 3MP self-pair and the destabilization of
the 3MP:dG mispair results in a self-pair thermal selectivity
Figure 1. Non-natural nucleobases. a) Pyridone analogues, b) previ-
[
10]
[
2a,4]
of 2–78C, which approaches that of a natural base pair.
ously reported ICS analogues,
and c) previously reported carbocy-
[
9,11]
The polymerase-mediated synthesis of these pyridone
self-pairs was characterized by incorporating the analogues
into template DNA and measuring the rate at which Kf
incorporates the non-natural triphosphates (Table 2). Self-
pair synthesis was inefficient, with second-order rate con-
clic analogues.
3MP=3-methylpyridone, 4MP=4-methylpyridone,
5
MP=5-methylpyridone, PYR=pyridone.
The b-nucleotides, triphosphates, and phosphoramidites
of the pyridone analogues were synthesized by a modification
[
8]
3
3
À1
À1
of a literature procedure as described in the Supporting
Information. To evaluate the thermodynamic stability of the
non-natural base pairs in duplex DNA, each non-natural
nucleoside was incorporated into complementary oligonu-
stants (k /K ) varying between < 10 and 4 ” 10 m min
cat M
(for comparison, a natural base pair is synthesized in a similar
7
À1
À1 [4]
sequence context with a rate constant of 5 ” 10 m min
).
The BEN self-pair (Figure 1) was synthesized with a similar
[
11]
cleotides and the melting temperature (T ) of the resulting
rate,
suggesting that the N-glycoside moiety does not
m
duplexes was determined by thermal denaturation experi-
ments (Table 1). The non-natural self-pairs are generally
more stable than mispairs formed with natural nucleotides,
with Tm values that range from 49.3 to 53.48C. With the
exception of 3MP, which is 38C more stable than the other
facilitate non-natural base pair synthesis, at least not within
the simple pyridone scaffold. The rates for pyridone self-pair
synthesis are inefficient in contrast with self-pairs formed
between other N-glycoside analogues bearing larger aromatic
surface areas, such as ICS and PICS (Figure 1b), which are
synthesized with second-order rate constants of approxi-
self-pairs, the T values are only slightly dependent on methyl
m
5
À1
À1 [2a,4]
substitution. The relative stability of the 3MP self-pair likely
reflects optimized packing of the 3-position methyl groups in
the interbase interface. Also, with the exception of 3MP,
these pyridone self-pairs are less stable than the self-pairs of
mately 10 m min .
Perhaps the intercalation of the large
Table 2: Steady-state rate constants of Kf-mediated incorporation of
[
a]
dXTP with dX in the template.
[
9]
the corresponding carbocyclic analogues (Figure 1c). This
relative destabilization may result from reduced hydropho-
bicity and/or from electrostatic repulsion between the car-
bonyl groups in the minor groove.
5
’-dTAATACGACTCACTATAGGGAGA
3’-dATTATGCTGAGTGATATCCCTCTXGCTAGGTTACGGCAGGATCGC
À1
À1
À1
X
kcat [min
]
K
M
[mm]
k
cat/K
[m min
]
M
3
3
MP
0.15Æ0.01
1.1Æ0.2
76Æ23
2.0”10
2.8”10
3
3
To examine the thermal orthogonality of the non-natural
base pairs, we determined the duplex melting temperatures
with the non-natural nucleotides paired opposite each natural
nucleotide (Table 1). These mispairs are significantly less
stable than even the least-stable self-pair, with the exception
4MP
5MP
PYR
389Æ96
135Æ65
0.53Æ0.18
3.9”10
[
b]
[b]
3
n.d.
n.d.
<10
[a] For experimental details, see the Supporting Information. [b] Rate too
slow to determine kcat and KM independently.
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Communications
aromatic rings reorients the pyridone carboxy groups or
provides sufficient stabilization in the transition state to
overcome their forced desolvation.
efficient extension is of great importance. To determine how
the pyridone and methyl modifications impact this critical
step, we examined the efficiencies with which Kf extended
primers (by incorporation of the next correct triphosphate,
deoxycytidine triphosphate; dCTP) that terminated with the
self-pairs (Table 3). Both the 5MP and PYR self-pairs were
To explore synthesis as a function of methyl group
substitution, we examined the synthesis of the 12 possible
heteropairs (see the Supporting Information). Although most
heteropairs are synthesized only inefficiently (with k /K
cat
M
3
3
À1
À1
Table 3: Selected rate constants for Kf-mediated extension of non-
between < 10 and 6 ” 10 m min ), the triphosphate of 4MP
is inserted opposite 3MP with an efficiency of 2 ”
0 m min , which is at least fivefold more efficient than
[
a]
natural termini by incorporation of dCTP.
4
À1
À1
1
5’-dTAATACGACTCACTATAGGGAGAX
3
’-dATTATGCTGAGTGATATCCCTCTYGCTAGGTTACGGCAGGATCGC
either self-pair. The increased rate results largely from an
À1
À1
À1
X
Y
kcat [min
]
KM [mm]
kcat/KM [m min
]
increased k , suggesting that the interbase interface is
cat
3
stabilized by packing interactions between the methyl
groups in the developing transition state, and, importantly,
that the pyridone scaffold may be optimized for synthesis.
To examine the kinetic orthogonality against pairing with
the natural bases, we characterized the ability of the pyridone
nucleobases to direct Kf to incorporate the natural deoxyri-
bonucleotide triphosphates (dNTPs; see the Supporting
Information). All of the mispairs are formed with a second-
3MP
3MP
4MP
5MP
PYR
3MP
4MP
3MP
4MP
1.1Æ0.1
2.9Æ0.9
7.3Æ0.7
9.6Æ1.0
13Æ4
270Æ107
202Æ56
107Æ12
117Æ32
198Æ21
204Æ56
322Æ76
146Æ56
3.9”10
4
4
5
MP
MP
1.5”10
4
6.8”10
4
PYR
8.2”10
4
5
5
MP
MP
6.4”10
4
16Æ3
7.8”10
4
PYR
PYR
14Æ2
4.3”10
5
16Æ4
1.1”10
[a] For experimental details and additional heteropair extension rate
4
À1
À1
order rate constant of less than 5 ” 10 m min . The most
efficiently synthesized mispairs resulted from insertion of
deoxyguanosine triphosphate (GTP), which was inserted
against the pyridone analogues significantly faster than
against the corresponding carbocyclic analogues. For the
carbocyclic analogues, each templates dGTP misincorpora-
constants, see the Supporting Information.
4
extended relatively efficiently at rates of 7 ” 10 and 8 ”
4
À1
À1
10 m min , respectively, which is approximately 50-fold
[
11]
faster than the BEN self-pair. The 4MP and 3MP self-pairs
4
were extended less efficiently, at rates of 1 ” 10 and 4 ”
3
À1
À1 [11]
3
À1
À1
tion at rates less than 2 ” 10 m min .
Methyl group
10 m min . All of the pyridone self-pairs are extended
substitution appears to have little effect on dGTP misincor-
poration, thus, it seems likely that the relatively efficient
synthesis of the mispair is driven by a H-bond formed
between the pyridone keto group and the dGTP amine.
After dGTP, the next most efficiently inserted natural
triphosphate was generally deoxyadenosine triphosphate
significantly more efficiently than their carbocyclic ana-
[
11]
logues.
This is largely due to increased kcat values. The
large difference in kcat between PYR and BEN self-pair
extension suggests that the minor-groove H-bond is important
for mediating interactions in the developing transition state.
This agrees with previous studies that show that removing the
(
dATP), which was inserted with a second-order rate constant
minor groove H-bond acceptor from dG at a primer terminus
3
4
À1
À1
[13]
ranging from 4 ” 10 –2 ” 10 m min , with the exception of
MP, which did not template dATP insertion at a detectable
results in a specific decrease in the extension k . As was
pol
[
6b]
3
suggested for the extension of a natural base pair, it seems
likely that the pyridone minor groove H-bond acceptor
stabilizes the rate-limiting transition state by engaging in a H-
bonding network with Arg668 of Kf and the ribosyl oxygen of
the incoming dNTP.
3
À1
À1
rate (k /K < 10 m min ). In contrast, the fidelity of the
carbocyclic analogues is typically limited by facile insertion of
dATP (often with rates in excess of 10 m min
cat
M
5
À1
À1 [11]
). This
data further supports the previous suggestion that hydro-
phobic minor-groove functional groups increase the efficiency
of dATP incorporation, possibly through a favorable packing
interaction with the methyne group of dATP. Furthermore,
the pyridones are more pyrimidine-like than their carbocyclic
counterparts, and their mispairs with dA may be better
recognized by the polymerase which evolved to discriminate
against natural mispairs.
The misincorporation of deoxythymidine triphosphate
(
to the carbocyclic analogues, inefficient incorporation of
dTTP opposite the pyridone analogues generally appears to
be due to weaker apparent binding of the natural triphos-
phate, again possibly reflecting forced desolvation of the
minor-groove carbonyl groups in the transition state. dCTP
was not incorporated opposite any pyridone analogue at a
detectable rate (k /K < 10 m min ).
As discussed above, the replication of predominantly
hydrophobic non-natural base pairs is consistently limited by
To further explore extension as a function of methyl group
substitution, we characterized the rates at which Kf extended
a primer terminating at a heteropair (X:Y, in which X is the
primer nucleobase and Y is the template nucleobase, Table 3
and the Supporting Information). Heteropairs formed
between either 3MP and 5MP or 3MP and PYR are
[
12]
4
4
À1
À1
extended at rates ranging from 4 ” 10 to 9 ” 10 m min .
These rates are 10- to 30-fold more efficient than those for the
corresponding self-pairs and are similar to the extension of
the 5MP and PYR self-pairs. Likewise, heteropairs formed
between 4MP and either 5MP or PYR were extended
significantly faster than the 4MP self-pair with rates ranging
3
À1
À1
dTTP) was inefficient (k /K < 5 ” 10 m min ). Relative
cat
M
11]
[
4
5
À1
À1
from 8 ” 10 to 2 ” 10 m min . This suggests that steric
interactions are responsible for the inefficient extension of
the 3MP and 4MP self-pairs, and that shape complementarity
is important for extension and possibly for the correct
positioning of the minor groove H-bond acceptor to produc-
tively engage the polymerase. It is also interesting to note that
the 4MP:5MP, PYR:4MP, and PYR:5MP heteropairs are
3
À1
À1
cat
M
[
5,11]
their extension.
Thus, understanding determinants of
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Angewandte
Chemie
extended within twofold of the most efficiently extended non-
natural base pairs reported to date.
[9] S. Matsuda, F. E. Romesberg, J. Am. Chem. Soc. 2004, 126,
4419 – 14427.
10] M. Berger, A. K. Ogawa, D. L. McMinn, Y. Wu, P. G. Schultz,
F. E. Romesberg, Angew. Chem. 2000, 112, 3069 – 3071; Angew.
Chem. Int. Ed. 2000, 39, 2940 – 2942.
1
[
[
The data have several important implications for future
efforts to design non-natural base pairs. Generally, at least for
the pyridone scaffold, shape complementarity is less impor-
tant for mispair stability and synthesis than is H-bonding.
However, shape complementarity between the nucleobase
analogues does appear to be important for base pair synthesis
and extension. It may contribute to efficient synthesis by
forming a well-packed interbase interface that stabilizes the
developing transition state. It may contribute to efficient
extension by contributing to the formation of a primer
terminus that properly orients the minor-groove H-bond
acceptor for polymerase recognition. The effects of shape
complementarity were generally less pronounced with pairs
formed between larger nucleobase analogues, likely owing to
11] S. Matsuda, A. A. Henry, F. E. Romesberg, J. Am. Chem. Soc.
2006, 128, 6369 – 6375.
[12] K. Kincaid, J. Beckman, A. Zivkovic, R. L. Halcomb, J. W.
Engels, R. D. Kuchta, Nucleic Acids Res. 2005, 33, 2620 – 2628.
13] T. E. Spratt, Biochemistry 2001, 40, 2647 – 2652.
14] a) A. K. Ogawa, Y. Wu, M. Berger, P. G. Schultz, F. E. Romes-
berg, J. Am. Chem. Soc. 2000, 122, 8803 – 8804; b) C. Yu, A. A.
Henry, F. E. Romesberg, P. G. Schultz, Angew. Chem. 2002, 114,
[
[
3997 – 4000; Angew. Chem. Int. Ed. 2002, 41, 3841 – 3844.
[15] A. M. Leconte, L. Chen, F. E. Romesberg, J. Am. Chem. Soc.
2005, 127, 12470 – 12471.
[
2a,4]
their intercalative mode of interaction.
The data also show
that, compared to the fully carbocyclic analogues, the
pyridone base pairs are generally extended with dramatically
increased rates and are also more orthogonal to the natural
[
11]
nucleotides, especially dA. In contrast with other classes of
predominantly hydrophobic nucleobase analogues, non-nat-
ural base pairs formed between the pyridone analogues
appear to be limited by their synthesis efficiencies. However,
the relative efficiency with which the 4MP:3MP pair is
synthesized suggests that these rates might be increased by
nucleobase optimization, as has been observed for other
[
2b,14]
scaffolds.
In addition, the synthesis might be optimized
through the directed evolution of polymerases to more
efficiently catalyze this step as it has for the PICS self-
[
15]
pair. Thus, further exploring these non-natural base-pair
scaffolds and evolving polymerases that more efficiently
synthesize them represents a promising strategy for the
expansion of the genetic alphabet.
Received: March 31, 2006
Published online: May 30, 2006
Keywords: DNA replication · genetic alphabet ·
.
hydrophobic effect · nucleobases · pyridones
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