Base-pairing properties of a structural isomer of glycerol nucleic acid

Article abstract of DOI:10.1002/anie.201300795

Know your limit! IsoGNA (a structural isomer of GNA) was found - in sharp contrast to GNA - to be highly restricted in its ability to base-pair with itself and other nucleic acids. While homogeneous sequences (e.g. isoGNA(A) ₁₆) formed duplexes, the heterogeneous sequences showed no base-pairing. This exemplifies the limitations of canonical nucleobases as the recognition elements in simpler, more primitive phosphate backbones. Copyright

Full text of DOI:10.1002/anie.201300795

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DOI: 10.1002/anie.201300795
Modified Nucleic Acids
Base-Pairing Properties of a Structural Isomer of Glycerol Nucleic
Acid**
Phaneendrasai Karri, Venkateshwarlu Punna, Keunsoo Kim, and
Ramanarayanan Krishnamurthy*
The investigation of constitutionally simple oligomeric infor-
mation systems is needed from the viewpoint of knowing how
structurally simple, and minimal, an oligomeric system can
become and still be functional (with respect to information
storage and processing). Herein, we report on the synthesis
and investigation of the base-pairing properties of one such
system, a glycerol-(3’!1’)-linked oligonucleotide.
Our interest in minimal, oligomeric information systems
was motivated by the experimental mapping of potentially
primordial oligomeric information systems,[1] which contain
Figure 1. Derivation of the 3’!1’-glycerolphosphate linked oligonucleo-
tide based on qualitative conformational analysis[3] of an idealized
A-form RNA/DNA backbone.
backbones, recognition elements, and linker groups that are
structurally quite different from conventional nucleic acids,
an approach pioneered by Eschenmoser and co-workers.[2] In
this pursuit, we used a “qualitative conformational analysis”[3]
approach—a simple analysis of the capability of the oligo-
meric system to adopt regular and repetitive conformations,
which is a prerequisite for base-pairing. Such a method can
also be used to design novel synthetic oligomeric structures.[3]
Employing this approach, we derived from the A-form of
DNA and RNA, a glycerol based acyclic-(3’!1’)-linked
oligonucleotide system that is tagged at the 2’-position with
canonical nucleobases (Figure 1).
This acyclic oligomer (Figure 2c) shows a resemblance to
the first acyclic system—glycerol-derived acyclic nucleo-
sides—which was considered by Joyce et al.[4a] when making
the case for an ancestral genetic system involving simpler
acyclic analogues of RNA and DNA (Figure 2a). Subse-
quently, this acyclic system (FNA) was shown to have very
weak base-pairing abilities.[5] Later, Meggers and co-work-
ers[6] showed that GNA,[7] in spite of having an acyclic
backbone, had strong base-pairing properties (Figure 2b).
This led to further study of acyclic nucleic acids.[8] Our
glycerol-based system, depicted in Figure 1 and Figure 2, is
a structural isomer of GNA (isoGNA)—with the nucleobase
at the 2’-position (instead of the 1’-position as in GNA) and
has 3’!1’-linked phosphodiester bonds (instead of the 3’!2’
Figure 2. Comparison of the three, flexible, acyclic glycerol-backbone-
based oligonucleotides.
linkages in GNA). When compared to FNA,[4,5] our system
lacks the oxymethylene linker between the nucleobase and
the backbone—placing the recognition element directly on
the backbone; however at the monomeric level, the backbone
units of both systems are achiral, unlike GNA. Therefore, we
investigated the base-pairing potential of isoGNA, which we
considered to have a structure that is in between that of FNA
and GNA.
The appropriate isoGNA phosphoramidites 6a–d
(Figure 3) were synthesized starting from the commercially
available (s)-solketal (Supporting Information, Scheme S1).
The synthesis of isoGNA oligonucleotides followed standard
methods from the literature with some adjustments
(Table S3). IsoGNA (ig) oligomers with one end-protecting
group seemed to be stable under the conditions investigated,
and all our sequences had a 1’-O- (and/or 3’-O-) cap, either in
the form of a phosphate or a DNA or RNA nucleotide. Base-
pairing properties of the isoGNA sequences (Table S4) were
investigated by temperature dependent UV absorption and
circular dichroism (CD) spectroscopy in phosphate buffer
[*] Dr. P. Karri, Dr. V. Punna, Dr. K. Kim, Prof. Dr. R. Krishnamurthy
Department of Chemistry, The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: rkrishna@scripps.edu
Homepage: http://www.scripps.edu/krishnamurthy/
[**] This work was supported by NASA Astrobiology: Exobiology and
Evolutionary Biology Program (Grant NNX09AM96G), and was
jointly supported by NSF and NASA Astrobiology Program under
the NSF Center for Chemical Evolution, Grant CHE-1004570. We
thank Professors A. Eschenmoser and G. Joyce, and Dr. Matthias
Stoop for constructive feedback on the manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201300795.
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Surprisingly, the heterobase-containing sequences
(isoGNA-(A,T)) showed no base-pairing. Both alternating
sequences ig(TA)8 and ig(T8A8) showed no sigmoidal behavior
in their UV-melting curves. Moreover, no duplex formation
was found for any of the irregular mixed isoGNA-(A,T)
16-mer sequences investigated (Entries 10–11 in Table S4).
Mixed 8-mer as well as 16-mer sequences containing A, T, G,
and C residues also showed no base-pairing (UV- and CD-
measurements). Such contrasting behavior where the homo-
geneous sequences show base-pairing but the heterogeneous
sequences do not is unprecedented within a given backbone
system.
Figure 3. The nucleobase-modified glycerol phosphoroamidites (6)
used in our study. DMTr=dimethoxytrityl; B=nucleobase; DPC=
diphenylcarbamoyl.
i
(pH 7.0, 1m NaCl). Initially, we studied g(T)12 and observed
no cooperative melting behavior with corresponding comple-
mentary RNA or DNA sequences. Therefore, we decided to
investigate the properties of hexadecamers of isoGNA.
The base-pairing behavior of isoGNA with the corre-
sponding complementary RNA and DNA sequences was,
once again, in contrast with GNA. For example, the homo-
i
i
No self-association was found for g(T)16; not unexpect-
purinic g(A)16 oligomer base-paired with both complemen-
i
edly, g(A)16 showed weak self-pairing,[9] which was sup-
tary DNA and RNA sequences (Figure 5; Entries 1–6 in
Table S5). This is in contrast to GNA, where there is
interaction with RNA but not with DNA. In the isoGNA
series, the RNA backbone also seems to be preferred over
a DNA backbone (Figure 5a). A 1:1 stoichiometry of duplex
formation was indicated by a job plot (Figure S5), although
the formation of a higher order complex (2:1) was also found
during the mixing experiment. There is almost no hysteresis
for these pairings with DNA or RNA, contrary to the curves
from self-pairing of isoGNA (Figure S4). This indicates that
the nature of the phosphate backbone dictates the kinetics of
duplex formation. The inverse combination, the dT-ig(T)16-dT
with d-(A)18 (or poly-dA) showed no duplex formation (as in
planted by reasonably strong heteroduplex formation in the
presence of complementary ig(T)16 (Figure 4; Figure S3).
Figure 4. Thermal melting curves of duplexes formed from dA-ig(A)16-
dA and dT-ig(T)16-dT oligonucleotides measured by UV spectroscopy;
ig=isoGNA; d=DNA; h1=reheating of a sample (after the first
measurement) kept at 48C for 6 days; c=cooling of the same sample;
h2=second heating cycle (after waiting at 48C for 1 min); %H=per-
cent hyperchromicity. Conditions: 5 mm of each oligonucleotide, 1m
NaCl, 10 mm Na2HPO4, 0.1 mm Na2EDTA, pH 7.0.
However, the hybridization behavior showed strong depend-
ence on time. A freshly prepared sample showed triplex-like
behavior. The lower transition was attributed to self-associ-
i
ation of g(A)16—a kinetically preferred process over the
i
thermodynamically more stable mixed duplex g(A)16/ig(T)16.
This was confirmed when the sample was allowed to
equilibrate at 48C for six days; only a single melting-
temperature (Tm) transition (h1 in Figure 4) was seen for
i
i
the duplex between g(A)16 and g(T)16, indicating that the
kinetics of its formation is relatively slow. Variation in the Tm
values with a change in the oligomer and salt concentrations
or replacement of NaCl with MgCl2, corroborated the
formation of interstrand duplexes. The corresponding tem-
perature dependent CD-spectrum showed weak signals
indicating that the duplex has little secondary structure
(Figure S3). This CD-behavior (and duplex stability) was
independent of the nature of the final capping residue on the
isoGNA oligomer (RNA, DNA, or free OH group).
Figure 5. Base-pairing behavior of a duplex formed by dA-ig(A)16-dA
with complementary DNA (d=DNA) and RNA (r=RNA). a) Thermal
melting curves; for conditions see footnote of Table S5. b) Temperature
dependent CD spectra; 5 mm of each oligonucleotide, 1m NaCl, 10 mm
Na2HPO4, 0.1 mm Na2EDTA, pH 7.0.
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FNA).[5] Once again, none of the heterogeneous sequences of
isoGNA, containing any combination of A, T, G, and C
residues, formed a duplex with the corresponding comple-
mentary RNA or DNA sequences.
The fact that ig(T)n sequences pair with ig(A)n but not with
complementary homopurinic sequences in the DNA and
RNA series, suggested that the isoGNA backbone may have
a preference for acyclic backbones, a phenomenon that could
be related to the flexibility of the backbone and inclination
between base-pairs on the backbone.[1d,10,11] Therefore, we
investigated the duplex formation of isoGNA sequences with
complementary alternative nucleobases on various acyclic
dipeptide backbones that were investigated previously in our
laboratory[1a–c] (Figure 6; Entries 10–16 in Table S5).
Figure 7. Thermal melting curves showing the duplex formation of
a) dT-ig(T)12-dT and dT-ig(T)16-dT with the corresponding 2,4-diamino-
1,3,5-triazine modified AspGlu-dipeptide backbone; b) dA-ig(A)16-dA
with a 16-mer 2,4-dioxo-5-(acylamino)pyrimidine modified AspGlu-
dipeptide backbone at 280 nm. Conditions: 5 mm of each oligonucleo-
tide, 1m NaCl, 10 mm Na2HPO4, 0.1 mm Na2EDTA, pH 7.0.
interference from the self-association of ig(A)16 was observed
(Figure S6). However, at 280 nm, only the melting of the
duplex could be observed (Figure 7b). In all of these acyclic
backbone intra-system pairings, little or no hysteresis was
observed, in contrast to the intra-system isoGNA pairings.
The interaction of GNAwith isoGNAwas examined using
two heterogeneous GNA sequences CACATTATTGTTGTA
and TACAACAATAATGTG,[13] and no base-pair mediated
interaction was seen. No interaction between isoGNA and
complementary homogeneous PNA sequences was observed
(Table S5), in contrast to acyclic dipeptide backbones with
alternative heterocycles (Figure 6 and Figure 7).
The striking difference in the intra- and inter-system base-
pairing of isoGNA, in combination with other studies,[4–6,14–16]
reinforces the notion that the backbone plays a critical role in
fine-tuning the nature of the base-pairing for the same set of
canonical nucleobases (found in nature). The acyclic nucleic
acid (FNA) system exhibits no intra-system base-pairing and
shows very limited duplex formation with RNA and DNA.[5]
GNA shows strong self-pairing but limited hybridization with
RNA and none with DNA,[6] and, isoGNA self- and cross-
pairing seems to be limited to homogeneous base sequen-
ces.[17,18] A shift in the position of the nucleobase in the acyclic
backbone (from the 1’- to the 2’-position), or variation in the
linker length (Figure 2), results in a drastic variation of base-
pairing capabilities, even though these systems contain the
same canonical nucleobases and closely related backbones.
Figure 6. Inter-system base-pairing of ig(A)16 and ig(T)16 with 2,4-
diamino-1,3,5-triazine, 2,4-dioxo-5-(acylamino)pyrimidine and 2,4-
diaminopyrimidine-6-carboxamide on acyclic dipeptide backbones.
Moderate to strong duplex formation was observed when
ig(T)n was mixed with the corresponding complementary 12-
and 16-mer 2,4-diamino-1,3,5-triazines (TNN) on a peptide
Asp-Glu backbone (Figure 7a). A clear dependence on the
length of the oligomer in the Tm confirmed an interstrand
association. The fact that even a 12-mer oligonucleotide
showed duplex formation—while this was not seen at the
same length for isoGNA with DNA or RNA—suggests the
important roles the nature of backbone, the orientation of the
hydrogen-bonding face, and charges in the backbone play in
determining ability to form a duplex.[12] Weak duplex
i
formation between g(T)16 and a 16-mer of 2,4-diaminopyr-
imidine-6-carboxamide on
a Asp-amAla backbone was
i
observed (Table S5). In the reverse combination, g(A)16 was
also found to form a duplex with a 2,4-dioxopyrimidine
modified Asp-Glu backbone (Figure 7b). At 260 nm and
below, the association of the heteroduplex along with
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there may be other primor-
dial recognition elements
that could function in con-
junction with simple, prebi-
otic backbones.[1] Thus, the
search for—and mapping
of—primordial information
systems could encompass
a wider variety of constitu-
ents, which are compatible
with heterotrophic and/or
Figure 8. Hypothesis to account for the unexpected dichotomous behavior between the homo- and
heterogeneous nucleobase sequences of isoGNA (arrows indicate the Watson–Crick base-pairing face). In the
homogeneous sequences, an all anti-purine (a) or an all syn-pyrimidine (b) arrangement (or the other way
around) allows for hydrogen-bond-mediated base-pairing. In a mixed sequence (c), the Watson–Crick faces
are pointing in different directions, thus hindering hydrogen-bond mediated associations.[19]
autotrophic
environ-
ments.[2,23]
Received: January 29, 2013
Revised: March 19, 2013
Published online: April 18, 2013
Keywords: acyclic nucleic acids · DNA · glycerol nucleic acid ·
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oligonucleotides · RNA
To account for the unexpected lack of base-pairing
between heterogeneous isoGNA sequences, we propose that
the purine and pyrimidine bases have different syn- and anti-
orientations (or the other way around) with respect to the
backbone (Figure 8).[18] Such different orientations would
allow the homo-sequences to form base-pairs because the
acceptor face of the pyrimidine from one strand would be
exposed to the donor face of the purine base. However, if they
are in the same (heterogeneous) sequence, then the hydro-
gen-bonding faces of the pyrimidine and purine bases would
be oriented in different directions, and one of the bases would
have to flip from the syn to the anti conformation (or the
other way around) to bring together uniform faces for base-
pairing.[19] If the energy gained by duplex formation is not
greater than the energy needed for such a change in base
orientation, then duplex formation will not be favored.
Such loss-of-base-pairing capability is also observed in
GNA with mixed sequences when paired with RNA. While
GNA A,T-sequences form duplexes with RNA, adding G and
C residues to the sequence causes GNA to lose this ability.[6b]
These results, when combined with ours, show that the nature
of the backbone is critical for determining, and limiting, the
recognition elements and the modes of base-pairing that can
be mediated by the canonical nucleobases. These observa-
tions caution against extrapolating base-pairing expectations
simply based on the behavior of homogeneous base sequen-
ces, at least for acyclic backbones.
[1] a) G. K. Mittapalli, K. R. Reddy, H. Xiong, O. Munoz, B. Han, F.
De Riccardis, R. Krishnamurthy, A. Eschenmoser, Angew.
Chem. 2007, 119, 2522; Angew. Chem. Int. Ed. 2007, 46, 2470;
b) G. K. Mittapalli, Y. M. Osornio, M. Guerrero, K. R. Reddy, R.
Krishnamurthy, A. Eschenmoser, Angew. Chem. 2007, 119, 2530;
Angew. Chem. Int. Ed. 2007, 46, 2478; c) X. Zhang, R.
Krishnamurthy, Angew. Chem. 2009, 121, 8268; Angew. Chem.
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[2] a) A. Eschenmoser, Origins Life Evol. Biosphere 2004, 34, 277;
b) A. Eschenmoser, Tetrahedron 2007, 63, 12821.
[3] A. Eschenmoser, Chimia 2005, 59, 836.
[4] a) G. F. Joyce, A. W. Schwartz, S. L. Miller, L. E. Orgel, Proc.
Natl. Acad. Sci. USA 1987, 84, 4398; b) M. Tohidi, L. E. Orgel, J.
Mol. Evol. 1989, 28, 367.
[5] a) K. C. Schneider, S. A. Benner, J. Am. Chem. Soc. 1990, 112,
453; b) F. Vandendriessche, K. Augustyns, A. Van Aerschot, R.
Busson, J. Hoogmartens, P. Herdewijn, Tetrahedron 1993, 49,
7223; c) P. Nielsen, L. H. Dreiøe, J. Wengel, Bioorg. Med. Chem.
1995, 3, 19; d) Y. Merle, E. Bonneil, L. Merle, J. Sagi, A. Szemzo,
Int. J. Biol. Macromol. 1995, 17, 239; e) B. M. Dahl, U.
Henriksen, O. Dahl, Org. Biomol. Chem. 2006, 4, 1115.
[6] a) L. Zhang, A. Peritz, E. Meggers, J. Am. Chem. Soc. 2005, 127,
4174; b) M. K. Schlegel, A. E. Peritz, K. Kittigowittana, L.
Zhang, E. Meggers, ChemBioChem 2007, 8, 927.
[7] GNAwas derived from threofuranosyl nucleic acid (TNA): K. U.
Schçning, P. Scholz, S. Guntha, X. Wu, R. Krishnamurthy, A.
Eschenmoser, Science 2000, 290, 1347.
[8] S. Zhang, C. Switzer, J. C. Chaput, Chem. Biodiversity 2010, 7,
245.
[9] For strong homo-adenine self-pairing see: J. Hunziker, H.-J.
Roth, M. Bçhringer, A. Giger, U. Diedrichsen, M. Gçbel, R.
Krishnan, B. Jaun, C. Leumann, A. Eschenmoser, Helv. Chim.
Acta 1993, 76, 259.
[10] H. Wippo, F. Reck, R. Kudick, M. Ramaseshan, G. Ceulemans,
M. Bolli, R. Krishnamurthy, A. Eschenmoser, Bioorg. Med.
Chem. 2001, 9, 2411.
[11] P. S. Pallan, P. Lubini, M. Bolli, M. Egli, Nucleic Acids Res. 2007,
35, 6611.
Overall, the base-pairing properties of the isoGNA
system stand in stark contrast to GNA and other flexible
nucleic acid backbones, necessitating discretion regarding the
structural requirements for a simple oligomeric information
system containing only the canonical nucleobases. This is
relevant to the evolution of information-containing poly-
mers—especially in the context of the origins of life.[4,20] The
search for structurally minimal (“simple”) informational
systems need not be constrained to the set of natural
nucleobases as the recognition elements. These canonical
nucleobases may represent a “functional optimum”[21] with
respect to the phosphate backbone, as the product of evolu-
tionary selection,[22] but this also allows for the possibility that
[12] GNA does not base-pair with TNA; see Y.-W. Yang, S. Zhang, E.
McCullum, J. C. Chaput, J. Mol. Evol. 2007, 65, 289.
[13] We are grateful to Prof. E. Meggers for providing these two
GNA sequences.
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[14] a) P. E. Nielsen, M. Egholm, R. H. Berg, O. Burchardt, Science
1991, 254, 1497; b) P. E. Nielsen, Origins Life Evol. Biospheres
1993, 23, 323.
[15] a) H. Asanuma, T. Toda, K. Murayama, X. Liang, H. Kashida, J.
Am. Chem. Soc. 2010, 132, 14702; b) H. Kashida, K. Murayama,
T. Toda, H. Asanuma, Angew. Chem. 2011, 123, 1321; Angew.
Chem. Int. Ed. 2011, 50, 1285.
interstrand phosphate distance (increased electrostatic repul-
sion).
[20] a) A. W. Schwartz, L. E. Orgel, Science 1985, 228, 585; b) A. W.
Schwartz, J. Visscher, C. G. Bakker, J. Niessen, Origins Life
Evol. Biospheres 1987, 17, 351; c) J. Visscher, A. W. Schwartz, J.
Mol. Evol. 1988, 28, 3; d) J. C. Chaput, C. Switzer, J. Mol. Evol.
2000, 51, 464.
[16] A. B. Petersen, M. A. Petersen, U. Henriksen, S. Hammerum, O.
Dahl, Org. Biomol. Chem. 2003, 1, 3293.
[17] An isoGNA-(G)n sequence was not studied owing to anticipated
complex associations.
[21] R. Krishnamurthy, Acc. Chem. Res. 2012, 45, 2035.
[22] It has been argued that the nucleobases in DNA have been
selected and optimized for DNA, from an ancestral RNA world:
A. C. Rios, Y. Tor, Astrobiology 2012, 12, 884.
[18] Collaborative studies are underway with Prof. N. Hud at Georgia
Institute of Technology (Atlanta) to understand this dichoto-
mous behavior.
[23] For a discussion see pages 12451 – 12453 and 12459 – 12462 in A.
Eschenmoser, Angew. Chem. 2011, 123, 12618; Angew. Chem.
Int. Ed. 2011, 50, 12412.
[19] Modeling studies suggest that other modes of base-pairing (e.g.
Hoogsteen) are disfavored because of the shortening of the
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