J. Am. Chem. Soc. 2000, 122, 8803-8804
8803
Rational Design of an Unnatural Base Pair with
Increased Kinetic Selectivity
Anthony K. Ogawa,† Yiqin Wu,† Markus Berger,
Peter G. Schultz,* and Floyd E. Romesberg*
Department of Chemistry, The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed April 26, 2000
In an effort to expand the genetic code we have examined a
variety of unnatural hydrophobic base pairs which pair selectively
in duplex DNA and are also synthesized efficiently by a DNA
polymerase.1-6 In some cases, the observed rates for the synthesis
of DNA containing unnatural base pairs approach those of natural
base pairs.2,4 However, the kinetic selectivity of the unnatural base
pairs has generally been limited by the fast enzymatic misincor-
poration of dATP opposite the hydrophobic bases in the template.
For example, as previously reported2 2MN, DMN, and TM
(Figure 1) each direct the exonuclease deficient Klenow fragment
of E. coli DNA polymerase I (KF) to incorporate dATP with an
efficiency (apparent kcat/KM) in excess of 105 M-1 min-1. The
“A-rule” proposes that dATP is preferentially inserted opposite
noninstructive sites in the template.7-9 This may imply that as a
result of the unnatural bases having none of the expected
H-bonding patterns, KF reads them in the template as “nonin-
stuctive”. An alternative to the “A-rule” for explaining the efficient
insertion of dATP is based on specific interactions between the
unnatural bases and the natural triphosphate. It is plausible that
the hydrophobic C2-methine of adenine (unique among the natural
bases) could interact favorably with the hydrophobic o-methyl
substituents of 2MN, DMN, and TM in the developing minor
groove. If this model is correct, deletion of the o-methyl
substituent could increase selectivity against dATP insertion and
result in more selective synthesis of the unnatural base pair.
To examine this model, 3MN and 2Np (Figure 1) were
synthesized and characterized. The 3MN and 2Np nucleosides
Figure 1. Unnatural hydrophobic bases.
were synthesized following a previously reported strategy (see
Supporting Information) and converted to the corresponding
triphosphates and phosphoramidites by literature methods.10,11 The
unnatural C-nucleosides were incorporated at position X of
oligonucleotide 5′-dATTATGCTGAGTGATATCCCTCTXGT-
CA to evaluate template-directed polymerase synthesis. We report
below that 3MN selectively enhances self-pair formation relative
to misincorporation of dATP. This leads to an unnatural base pair
that is enzymatically synthesized with an efficiency and fidelity
approaching those characteristic of native base pair synthesis.
Steady-state kinetic data corresponding to single nucleotide
incorporation are shown in Table 1.12 There is an 17-fold reduction
in incorporation efficiency for dATP opposite 3MN relative to
its methylated analogue DMN, while the kcat/KM for incorporation
of other native triphosphates opposite 3MN was largely unaf-
fected.2 The 3MN:3MN self-pair is efficiently synthesized (kcat/
KM ) 2.4 × 106 M-1 min-1) resulting in a 62-fold selectivity for
correct self-pair synthesis. Although DNA synthesis beyond the
3MN:3MN self-pair proceeded inefficiently, its highly selective
formation represents an important development in our efforts to
design a third base pair.
* To whom correspondence should be addressed: Telephone (858) 784-
7290, Fax (858) 784 7472, E-mail floyd@scripps.edu.
To deconvolute the impact of the m-methyl group, 2Np was
synthesized (Figure 1). Surprisingly, KF efficiently inserts dATP
opposite 2Np in the template. The differing behaviors of the
regioisomeric o- and m-methyl substitution may be rationalized
based on the specific interactions between the hydrophobic
template and dATP. This model hypothesizes that ortho substit-
uents favor the incorporation of dATP by providing hydrophobic
contacts with the incoming adenine nucleobase, while meta
substituents disfavor the KF mediated insertion of dATP due to
forced desolvation of the adenine hydrophilic endocyclic amide
moiety.2,4,13 The kinetic data support this model if rotational
isomerization about the C-glycosidic linkage is considered (Figure
2). TM is known to reside in the anti configuration about the
C-glycosidic linkage,14 and the rotation to the syn conformation
is disfavored due to the resulting eclipsing interactions between
the o-methyl group and the ribosyl oxygen lone pairs. The same
is likely true for 2MN and DMN. Therefore, 2MN, DMN, and
TM each present the incoming triphosphate nucleobase with an
o-methyl group disposed toward the developing minor groove.
† These authors contributed equally to this work.
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10.1021/ja001450u CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/26/2000