Polymerase recognition of 2-thio-iso-guanine·5-methyl-4-pyrimidinone (iGs·P) - A new DD/AA base pair

Article abstract of DOI:10.1016/j.bmcl.2016.01.041

Polymerase specificity is reported for a previously unknown base pair with a non-standard DD/AA hydrogen bonding pattern: 2-thio-iso-guanine·5-methyl-4-pyrimidinone. Our findings suggest that atomic substitution may provide a solution for low fidelity previously associated with enzymatic copying of iso-guanine.

Full text of DOI:10.1016/j.bmcl.2016.01.041

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1177–1179
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Polymerase recognition of 2-thio-iso-guanineꢀ5-methyl-4-
pyrimidinone (iGsꢀP)—A new DD/AA base pair
y
⇑
Dong-Kye Lee , Christopher Switzer
Department of Chemistry, University of California, Riverside, CA 92521, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Polymerase specificity is reported for a previously unknown base pair with a non-standard DD/AA hydro-
gen bonding pattern: 2-thio-iso-guanineꢀ5-methyl-4-pyrimidinone. Our findings suggest that atomic
substitution may provide a solution for low fidelity previously associated with enzymatic copying of
iso-guanine.
Received 8 January 2016
Revised 13 January 2016
Accepted 14 January 2016
Available online 16 January 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Synthetic biology
Origins of life
Nucleic acids
Base-pair
The distinct hydrogen-bonding patterns found in natural AꢀT
and GꢀC Watson–Crick base pairs enable the storage and transfer
of genetic information in all terran organisms. Over the past two-
and-a-half decades, considerable effort has been expended to
increase the number of functional Watson–Crick-like base pairs
using non-standard hydrogen bonding patterns, or other types of
non-covalent interactions. These efforts have led to non-standard
DNA base pairs capable of enzymatic replication in the context of
PCR and a semi-synthetic organism, as well as an enhanced under-
standing of the physical principles underlying both base-pairing
and replication.1,2
The iGꢀiC base-pair was postulated over fifty years ago as an
extension of the natural set of Watson–Crick hydrogen bonding
patterns.3 Subsequent assessment of this proposal showed that
the iGꢀiC pair was incompletely functional for replication and
translation owing to the formation of mispairs between iG and
T.4 The tendency of iG to pair with both iC and T has been attribu-
ted to the coexistence of N1–H and O2–H iG tautomers.5 Several
strategies have been investigated to date to improve the fidelity
of iGꢀiC, including the replacement of iG with isoinosine,6 7-
deaza-iG,7 7-halo-7-deaza-iG,8 8-aza-iG,9 and HNA-iG.10 While
several of the nucleobase analogs increased the N1–H tautomer
content of iG, none of the iG alternatives examined enable replica-
tion in the absence of high levels of iG ? A transitions.
Sulfur modified nucleobases occur naturally in tRNA, and
include 2-thio-U, 4-thio-U and 2-thio-C, among others.11 More-
over, the anticipated prebiotic accessibility of sulfur-modified
nucleobases suggests that they are fundamental constructs.12
Beyond tRNA, the effect of sulfur substitution on DNA has been
investigated, including: 2-S-T,13 4-S-T,13,14 2-S-C,15 and 6-S-G.16
As part of the continuing search for an iG replacement structure
that exhibits fidelity for its nonstandard pyrimidine complement,
we report on 2-thio-iG (iGs) interactions with 5-methyl-4-pyrim-
idinone (P) and iC, both in a DNA double helix and under the direc-
tion of a DNA polymerase, including faithful enzymatic copying of
the iGsꢀP base pair (Fig. 1).
Before conducting polymerase studies, the iGsꢀP and iGsꢀiC base-
pair and mismatch stabilities were assessed by UV-monitored ther-
mal denaturation of DNA double helices. Towards this end, we syn-
thesized complementary oligonucleotides containing the iGs, iG, P
and iC nonstandard nucleobases. iGs was synthesized and incorpo-
rated into DNA using the procedure we reported recently.17 DNA oli-
gomers bearing
P were synthesized from the corresponding
phosphoramidite, which was prepared as outlined by Rajur and
McLaughlin.18 Table 1 summarizes the results from the thermal
denaturation studies. Based on the two duplexes studied, the rela-
tive base-pair stabilities were observed on average as:
GꢀC ꢁ iGꢀiC > AꢀT > iGsꢀiC > iGꢀP. While it was not always possible
to detect mismatches in the case of the smaller nonamer duplex,
the mismatches that were detected along with those observed in
the dodecamer case revealed several notable features. First, iGs
exhibits a leveling of the C and T mismatch stabilities in comparison
⇑
Corresponding author. Tel.: +1 951 827 7266; fax: +1 951 827 4713.
E-mail address: switzer@ucr.edu (C. Switzer).
Present address: Department of Forensic Chemistry, Busan Institute, National
y
Forensic Service, Yangsan-si 626-742, Republic of Korea.
http://dx.doi.org/10.1016/j.bmcl.2016.01.041
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

1178
D.-K. Lee, C. Switzer / Bioorg. Med. Chem. Lett. 26 (2016) 1177–1179
Figure 1. The 2-thio-iso-guanineꢀ5-methyl-4-pyrimidinone (iGsꢀP) and iso-
guanineꢀ5-methyl-iso-cystosine (iGꢀiC) base-pairs.
to iG, which shows a greater affinity for T over C. Second, both P and
iC form stronger mispairs with G than A.
Polymerase studies were performed with the template/primer
combinations illustrated in Figure 2A in conjunction with AMV
reverse transcriptase previously employed for iG.4b Initially, the
ability was probed of template iGs to direct the incorporation
and extension of its intended cognate base, iC, while avoiding
Figure 2. (A) The polymerase catalyzed reaction studied, including primer and
template sequences. (B) Autoradiograph of the results of primer extension using
templates containing iGs or iG and only those dNTPs corresponding to the
nucleosides indicated above each lane, 9 U AMV reverse transcriptase, 37 °C, 1 h.
mispairing with
T or the other natural nucleobases. The
autoradiograph depicted in Figure 2B, obtained after PAGE
separation of the polymerase reaction products, clearly indicates
no template iGs directed incorporation of C, A, or G (lanes 2, 3,
5). Satisfyingly, iGs directed incorporation of T is nearly absent
(less than 1% based on densitometry, lane 4, Fig. 2B). In contrast
to the result in lane 4, when iGs is replaced by a template
bearing iG instead, complete incorporation of T is observed (lane
11, Fig. 2B) as expected based on prior studies due to the iG enol
tautomer.4,5 Problematically, however, iGs was found to no longer
direct the incorporation of iC (iGs lane 6 vs iG lane 12, Fig. 2B).
To circumvent the inability of iGs to direct the incorporation of
iC, we elected to remove the 2-amino group of the pyrimidine com-
ponent by replacing iC with P (Fig. 1). The outcome of this pyrim-
idine 2-substituent size reduction (NH2 ? H) was expected to be a
base pair possessing N9–N1 and C10–C10 distances identical to nat-
ural Watson–Crick pairs. Reactions catalyzed with AMV reverse
transcriptase exploring the replacement of diCTP with dPTP are
shown in Figure 3. As a point of reference, lane 5 in Figure 3 corre-
sponds to lane 4 of Figure 2B, demonstrating the nearly absent pair-
Figure 3. Autoradiograph of the results of primer extension using templates
containing iGs, A or G and only those dNTPs corresponding to the nucleosides
indicated above each lane, 9 U AMV reverse transcriptase, 37 °C, 1 h.
Table 1
Melting temperatures (Tm values) from UV monitored thermal denaturation of DNA
double helices. Samples contained 2.5 lM of each oligonucleotide strand, NaCl (1 M),
Na2HPO4 (10 mM, pH = 7.0), EDTA (0.1 mM, pH = 7.0). Errors in Tm values are
estimated as 0.5 °C
ing between iGs and T (less than 1% based on densitometry). In
contrast to the absence of incorporation of iC by iGs (lane 6,
Fig. 2B), ready incorporation of P by iGs is observed (lane 4, Fig. 3).
Following on the successful recognition of P by iGs, the absence of
P mispair formation with either A or G was probed and confirmed
(lanes 10 and 13, respectively, Fig. 3).
In summary, we have reported faithful polymerase copying of
the iGsꢀP base pair. In particular, we have shown polymerase direc-
ted incorporation by iGs of cognate pyrimidine P, and the absence
of incorporation of P by non-cognate purines A or G. Critically,
there is a near absence of polymerase directed misincorporation
by iGs of T—a fidelity issue that has plagued iG as a Watson–Crick
base pair component from its inception. On the basis of our results,
we suggest that iGsꢀP is a possible alternative to the iGꢀiC non-stan-
dard base pair. iGs appears to be unique among iG congeners in its
ability to avoid enzyme directed interactions with T. Efforts to fur-
ther explore the iGsꢀP base pair are in progress.

D.-K. Lee, C. Switzer / Bioorg. Med. Chem. Lett. 26 (2016) 1177–1179
1179
5. (a) Sepiol, J.; Kazimierczuk, Z.; Shugar, D. Z. Naturforsch 1975, 31, 361; (b)
Robinson, H.; Gao, Y.-G.; Bauer, C.; Roberts, C.; Switzer, C.; Wang, A. H. J.
Biochemistry 1998, 37, 10897.
6. (a) Beaussire, J.-J.; Pochet, S. Tetrahedron 1998, 54, 13547; (b) Beaussire, J.-J.;
Pochet, S. Nucleoside Nucleotides 1999, 18, 403.
7. (a) Martinot, T. A.; Benner, S. A. J. Org. Chem. 2004, 69, 3972; (b) Seela, F.; Peng,
X.; Xu, K. Nucleosides Nucleotides and Nucleic Acids. 2007, 26, 1569.
8. Seela, F.; Peng, X.; Li, H. J. Am. Chem. Soc. 2005, 127, 7739.
9. Jiang, D.; Seela, F. J. Am. Chem. Soc. 2010, 132, 4016.
Acknowledgments
This work was supported by the National Aeronautics and Space
Administration, Exobiology and Evolutionary Biology Program
NNX09AN01G), and the National Science Foundation CHE-
1310423).
10. Bande, O.; El Asrar, R. A.; Braddick, D.; Dumbre, S.; Pezo, V.; Schepers, G.;
Pinheiro, V. B.; Lescrinier, E.; Holliger, P.; Marliere, P.; Herdewijn, P. Chem. Eur.
J. 2015, 21, 5009.
Supplementary data
11. (a) Limbach, P. A.; Crain, P. F.; McCloskey, J. A. Nucl. Acids Res. 1994, 22, 2183;
(b) Kumar, R. K.; Davis, D. R. Nucl. Acids Res. 1997, 25, 1271; (c) Siegfried, N. A.;
Kierzek, R.; Bevilacqua, P. C. J. Am. Chem. Soc. 2010, 132, 5342; (d) Larsen, A. T.;
Fahrenbach, A. C.; Sheng, J.; Pian, J.; Szostak, J. W. Nucl. Acids Res. 2015, 43,
7675.
12. Robertson, M. P.; Levy, M.; Miller, S. L. J. Mol. Evol. 1996, 43, 543.
13. (a) Lezius, A. G. Eur. J. Biochem. 1970, 14, 154; (b) Faerber, P. Biochem. Biophys.
Res. Commun. 1973, 55, 873; (c) Connolly, B. A.; Newman, P. C. Nucl. Acids Res.
1989, 17, 4957; (d) Sintim, H. O.; Kool, E. T. J. Am. Chem. Soc. 2006, 128, 396.
14. Rao, T. V. S.; Haber, M. T.; Sayer, J.; Jerina, D. M. Bioorg. Med. Chem. Lett. 2000,
10, 907.
15. (a) Stornetta, A.; Angelov, T.; Guengerich, F. P.; Sturla, S. J. ChemBioChem 2013,
14, 1634; (b) Pugliese, K. M.; Gul, O. T.; Choi, Y.; Olsen, T. J.; Sims, P.; Collins, P.
G.; Weiss, G. A. J. Am. Chem. Soc. 2015, 137, 9587.
16. (a) Yoshida, S.; Yamada, M.; Masaki, S.; Saneyoshi, M. Cancer Res. 1979, 39,
3955; (b) Rappaport, H. P. Nucl. Acids Res. 1988, 16, 7253.
Supplementary data (spectra of synthetic compounds and
experimental procedures) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.01.
041.
References and notes
1. (a) Benner, S. A. Acc. Chem. Res. 2004, 37, 784; (b) Krueger, A. T.; Kool, E. T. Curr.
Opin. Chem. Biol. 2007, 11, 588; (c) Henry, A. A.; Romesberg, F. E. Curr. Opin.
Chem. Biol. 2003, 7, 727; (d) Hirao, I.; Kimoto, M.; Yamashige, R. Acc. Chem. Res.
2012, 45, 2055.
2. Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T. J.; Dai, N.; Foster, J. M.; Correa,
I. R.; Romesberg, F. E. Nature 2014, 509, 385.
3. Rich, A. On the Problems of Evolution and Biochemical Information Transfer. In
Horizons in Biochemistry; Kasha, M., Pullman, B., Eds.; Academic Press: New
York, 1962; pp 103–126.
17. Lee, D.; Switzer, C. Nucleosides Nucleotides Nucleic Acids 2015, 34, 424.
18. Rajur, S. B.; McLaughlin, L. W. Tetrahedron Lett. 1992, 33, 6081.
4. (a) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989, 111, 8322;
(b) Switzer, C. Y.; Moroney, S. E.; Benner, S. A. Biochemistry 1993, 32, 10489; (c)
Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A. Nature 1992, 356, 537.