Nonenzymatic oligomerization of RNA by TNA templates

Article abstract of DOI:10.1021/ol062368s

(Diagram presented) Cytosine TNA promotes nonenzymatic, template-directed oligomerization of complementary activated rGMP, leading to selective and efficient formation of RNA products. This process models "genetic takeover" of a pre-RNA by RNA.

Full text of DOI:10.1021/ol062368s

ORGANIC
LETTERS
2006
Vol. 8, No. 25
5809-5811
Nonenzymatic Oligomerization of RNA
by TNA Templates
Benjamin D. Heuberger and Christopher Switzer*
Department of Chemistry, UniVersity of California, RiVerside, California 92521
switzer@ucr.edu
Received September 26, 2006
ABSTRACT
Cytosine TNA promotes nonenzymatic, template-directed oligomerization of complementary activated rGMP, leading to selective and efficient
formation of RNA products. This process models “genetic takeover” of a pre-RNA by RNA.
Accurate transfer of genetic information is critical for the
survival of an organism. In a prebiotic environment, however,
the sophisticated enzymes that facilitate replication of modern
nucleic acids would have been absent. As a result, prebiotic
informational polymers need to access other, nonenzymatic,
modes of replication. Yet, to date, RNA monomer synthesis
via prebiotic model reactions has proven elusive, although
several milestones have been attained in the area of ribose
synthesis and stability.1 Thus, a pre-RNA may have aided
the transition from prebiotic materials to the RNA world.2
Figure 1. Structures of (a) DNA, (b) RNA, (c) TNA, and (d)
(L)-R-Threose nucleic acid,3 TNA, is a variant of natural
2-MeImpG.
nucleic acids wherein ribose is replaced by a four-carbon
sugar (Figure 1). TNA has the capacity to form stable duplex
structures with both DNA and RNA.3 Further, threose is
formed during condensation of simple aldehydes in prebiotic
model reactions leading to ribose and allose.1c,4 These
observations raise the possibility that TNA may have been
a precursor to RNA during molecular evolution.2,5 In this
(1) (a) Ricardo, A.; Carrigan, M. A.; Olcott, A. N.; Benner, S. A. Science
2004, 303, 196. (b) Springsteen, G.; Joyce, G. F. J. Am. Chem. Soc. 2004,
126, 9578. (c) Mu¨ller, D.; Pitsch, S.; Kittaka, A.; Wagner, E.; Wintner, C.
E.; Eschenmoser, A. HelV. Chim. Acta 1990, 73, 1410.
(2) Joyce, G. F. Nature 2002, 418, 214.
work, we address the fitness of TNA by exploring its ability
10.1021/ol062368s CCC: $33.50
© 2006 American Chemical Society
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to serve as a template for nonenzymatic transfer of genetic
information.6,7 Specifically, we report successful nonenzy-
matic oligomerization of RNA on a TNA template (Scheme
1), a process that models genetic takeover8 of a pre-RNA
by RNA.
Scheme 1. Nonenzymatic, Template-Directed Oligomerization
Reaction Conducted with Templates 1-3
Template 3 from Figure 2 shown; tC ) TNA-C, rG ) ribo-G,
all other residues ) DNA.
Three hairpin templates with varying numbers of TNA
residues were synthesized (Figure 2, left of panel) on an ABI-
394 oligonucleotide synthesizer and purified by 20% dena-
turing PAGE.9 Hairpin templates enable low concentrations
of template/primers to be used without interference of off-
template reactions.6f All three TNA-bearing templates were
Figure 2. Time course study of nonenzymatic oligomerization
reactions on 32P-end-labeled TNA bearing templates 1-3 (tC )
TNA-C, rG ) ribo-G, all other residues ) DNA). The extent of
oligomerization was assessed at 30 min, 1 h, 4 h, 12 h, 1 day, 3
days, and 10 days for each template (lanes 2-8, respectively). Lane
1 is the template only. Oligomerization conditions were: 1.2 M
NaCl, 200 mM 2,6-lutidine‚HCl pH 8.0, 0.5 µM template, 100 mM
2-MeImpG and 200 mM MgCl2, 5 °C. The oligomerization
procedure from refs 6f and 7d,h was followed.
(3) (a) Scho¨ning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy,
R.; Eschenmoser, A. Science 2000, 290, 1347. (b) Scho¨ning, K.-U.; Scholz,
P.; Wu, X. L.; Guntha, S.; Delgado, G.; Krishnamurthy, R.; Eschenmoser,
A. HelV. Chim. Acta 2002, 85, 4111. (c) Pallan, P. S.; Wilds, C. J.; Wawrzak,
Z.; Krishnamurthy, R.; Eschenmoser, A.; Egli, M. Angew. Chem., Int. Ed.
2003, 42, 5893.
(4) Pitsch, S.; Eschenmoser, A.; Gedulin, B.; Hui, S.; Arrhenius, G.
Origins Life EVol. Biosphere 1995, 25, 297.
(5) Herdewijn, P. Angew. Chem., Int. Ed. 2001, 40, 2249.
(6) For past work exploring nonenzymatic oligomerization on natural
nucleotide templates, see: (a) Inoue, T.; Orgel, L. E. J. Am. Chem. Soc.
1981, 103, 7666. (b) Joyce, G. F.; Visser, G. M.; VanBoeckel, C. A. A.;
VanBoom, J. H.; Orgel, L. E.; VanWestrenen, J. Nature 1984, 310, 602.
(c) Chen, C. B.; Inoue, T.; Orgel, L. E. J. Mol. Biol. 1985, 181, 271. (d)
Joyce, G. F.; Orgel, L. E. J. Mol. Biol. 1986, 188, 433. (e) Acevedo, O. L.;
Orgel, L. E. J. Mol. Biol. 1987, 197, 187. (f) Wu, T. F.; Orgel, L. E. J. Am.
Chem. Soc. 1992, 114, 317. (g) Hill, A. R., Jr.; Orgel, L. E.; Wu, T. F.
Origins Life EVol. Biosphere 1993, 23, 285. (h) Zielinski, M.; Kozlov, I.
A.; Orgel, L. E. HelV. Chim. Acta 2000, 83, 1678. (i) Kanavarioti, A.;
Bernasconi, C. F.; Baird, E. E. J. Am. Chem. Soc. 1998, 120, 8575. (j)
Kanavarioti, A.; Baird, E. E.; Hurley, T. B.; Carruthers, J. A.; Gango-
padhyay, S. J. Org. Chem. 1999, 64, 8323. (k) Kurz, M.; Gobel, K.; Hartel,
C.; Gobel, M. W. Angew. Chem., Int. Ed. Engl. 1997, 36, 842. (l) Hey, M.;
Hartel, C.; Gobel, M. W. HelV. Chim. Acta 2003, 86, 844.
(7) For past work exploring nonenzymatic oligomerization on nonstand-
ard nucleotide templates, see: (a) Rembold, H.; Robins, R. K.; Seela, F.;
Orgel, L. E. J. Mol. EVol. 1994, 38, 211. (b) Bohler, C.; Nielsen, P. E.;
Orgel, L. E. Nature 1995, 376, 578. (c) Ertem, G.; Ferris, J. P. Nature
1996, 379, 238. (d) Prakash, T. P.; Roberts, C.; Switzer, C. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 1522. (e) Kozlov, I. A.; Politis, P. K.; Pitsch, S.;
Herdewijn, P.; Orgel, L. E. J. Am. Chem. Soc. 1999, 121, 1108. (f) Kozlov,
I. A.; Orgel, L. E. HelV. Chim. Acta 1999, 82, 1799. (g) Chaput, J. C.;
Switzer, C. J. Am. Chem. Soc. 2000, 122, 12866. (h) Chaput, J. C.; Switzer,
C. J. Mol. EVol. 2000, 51, 464. (i) Kozlov, I. A.; Zielinski, M.; Allart, B.;
Kerremans, L.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.
Chem.-Eur. J. 2000, 6, 151. (j) Hartel, C.; Gobel, M. W. HelV. Chim.
Acta 2000, 83, 2541. (k) Chaput, J. C.; Sinha, S.; Switzer, C. Chem.
Commun. 2002, 15, 1568.
found to promote guanosine 5′-phosphoro-2-methylimidazole
(2-MeImpG) oligomerization. As Figure 2 shows, oligomer-
ization products are apparent after 3 days of incubation and
continue to form up to day 10. The rates of oligomerization
in Figure 2 depend on TNA content. Whereas oligomeriza-
tion on template 1 (one TNA residue) appears to be all but
complete after 3-5 days, templates 2 (two TNA residues)
and 3 (seven contiguous TNA residues) require 5-10 days
to achieve maximum oligomerization. Past work on hairpins
appended with (rC)7 and (dC)5 templates showed the half-
lives for disappearance of the templates to be 0.837d and 3.0
h,6f respectively, for the oligomerization of 2-MeImpG. By
plotting the disappearance of templates 1, 2, and 3 when
incubated in the presence of 2-MeImpG, we estimate the
half-lives as 14, 37, and 53 h, respectively.
Oligomerization products may contain 2′,5′- phosphodi-
ester linkages, 3′,5′-linkages, or a combination of the two.
To determine the types of linkages present in our system,
the reaction products from all three templates were treated
with RNase T1.6f,7d,h The RNase T1 enzyme cleaves oligori-
bonucleotides specifically at the 3′-side of a 3′,5′- linked
guanosine residue while leaving 2′,5′-linkages untouched.
Figure 3 shows that RNase T1 treatment of oligomerization
products from all three templates resulted in the cleavage of
the first linkage formed to give the template plus a 3′-
phosphate residue (comparison of lanes 1 and 4). Thus, the
(8) Cairns-Smith, A. G. Genetic TakeoVer and the Mineral Origins of
Life; Cambridge University Press: Cambridge, 1982.
(9) N4-Benzoyl-1-{2′-O-[(2-cyanoethoxy)(diisopropylamino)-phosphino]-
3′-O-[(4,4′-dimethoxytriphenyl)methyl]-R-L-threofuranosyl}-cytosine was
synthesized according to the reported procedure.3b Templates were synthe-
sized on an ABI 394 DNA synthesizer using standard protocols. Guanosine
5′-phosphoro-2-methyl-imidazolide was prepared from guanosine 5′-mono-
phosphate according to the reported procedure: Joyce, G. F.; Inoue, T.;
Orgel, L. E. J. Mol. Biol. 1984, 176, 279.
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Org. Lett., Vol. 8, No. 25, 2006

Figure 4. Evaluation of template-directed oligomerization fidelity
and divalent ion performance using template 3. Conditions: 10 day
reaction time, 1.2 M NaCl, 200 mM 2,6-lutidine‚HCl pH 8.0, 0.5
µM template 3, 100 mM 2-MeImpA (lane 1), 100 mM 2-MeImpG
(lanes 2-9), 5 °C. Divalent metal ion content [200 mM], lanes
1-9: MgCl2, no divalent ions, MgCl2, MnCl2, CoCl2, Ni(NO3)2,
ZnSO4, HgCl2, Pb(NO3)2.
may be attributed to nucleation of intrinsic 2-MeImpA
stacking at the hairpin template 3′-G terminus and subsequent
bond formation, possibly enhanced by weak interactions with
the template.
Effects of different divalent ions on oligomerization were
explored with template 3 incubated in the presence of
2-MeImpG. The results are shown in Figure 4, lanes 2-9.
Full-length oligomerization products were only observed
when the reaction contained MgCl2. All other divalent ions
used essentially led to no oligomerization.
Figure 3. RNase T1 digests of oligomerization products from
templates 1, 2, and 3 (panels a, b, and c, respectively). Lanes
contain: template only (lane 1), oligomerization products only (lane
2), oligomerization products and buffer with no enzyme (lane 3),
and oligomerization products, buffer, and RNase T1 (lane 4).
Our results demonstrate that TNA is capable of nonen-
zymatic, template-directed oligomerization of RNA, a neces-
sary capability of pre-RNA. In past work, PNA has been
investigated as a pre-RNA candidate, showing both a capacity
for formation from plausible prebiotic chemicals11 and
template-directed RNA oligomerization.7b,12 Although rates
of both PNA7b,12 and TNA template-directed oligomerization
are less than that of DNA or RNA templates, it is notable
that the 2-methylimidazole leaving group was optimized for
the latter polymers.13 Nevertheless, the data reported here
indicate that TNA rivals PNA in effectively promoting
template-directed synthesis of RNA. Further studies explor-
ing nonenzymatic replication with TNA are ongoing.
first phosphodiester bond formed during oligomerization on
all three templates is 3′,5′-linked.
Pyrophosphate byproducts may form when 2-MeImpG
reacts with the terminal 5′-phosphate instead of the terminal
2′/3′-hydroxyl group of a hairpin template. Pyrophosphate
byproducts may be revealed by treating the products from
an oligomerization reaction with calf intestinal alkaline
phosphatase (CIAP). In the assay, hairpin templates whose
5′-32P group has undergone pyrophosphate bond formation
with 2-MeImpG will be protected from CIAP and revealed
by subsequent PAGE autoradiographic analysis, whereas
those hairpin templates retaining a free 5′-32P group after
10 day incubation will have this group cleaved by CIAP and
be invisible to autoradiography. When the oligomerization
products from all three templates were treated with CIAP,
all product and template bands disappeared leaving very
minimal amounts of pyrophosphate byproducts to be ob-
served (data not shown).
Acknowledgment. Funding for this work from the
National Aeronautics and Space Administration is gratefully
acknowledged.
Supporting Information Available: Spectra of all syn-
thetic compounds and experimental procedures. This material
is available free of charge via the Internet at http://pubs.acs.org.
Although the above results demonstrate the viability of
TNA as a template for nonenzymatic oligomerization, base-
pairing fidelity is necessary for the transfer of genetic
information. Toward assessing the latter, 2-MeImpA was
incubated with template 3 for 10 days under the same
conditions as before. Minimal oligomerization was observed,
and no formation of full-length product was seen even after
a 10 day incubation period (Figure 4, lane 1). 5′-AMP is
known to associate via stacking interactions in the absence
of a template.10 The small extent of observed oligomerization
OL062368S
(11) Nelson, K. E.; Levy, M.; Miller, S. L. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 3868.
(12) Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E. Nucl. Acids Res. 1997,
25, 4797.
(13) Other factors appear superficially to favor TNA over DNA as a
template for RNA, including A-form structure3c,7i and association thermo-
dynamics of mixed base TNA/RNA sequences.3b However, TNA homoo-
ligomers of the general type used in the present study (e.g., tPyr/rPur) have
been shown to have anomalously low duplex stabilities.3b
(10) Morcillo, J.; Gallego, E.; Peral, F. J. Mol. Struct. 1987, 157, 353.
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