Kinetic analysis of hydrolytic reaction of homo- and heterochiral adenylyl(3′-5′)adenosine isomers: Breaking homochirality reduces hydrolytic stability of RNA

Article abstract of DOI:10.1039/b500673b

The hydrolytic stability of the diastereomeric isomers of ApA was compared and the results show that heterochiral ApAs are more rapidly hydrolyzed than homochiral ApAs at low temperatures, suggesting that hydrolytic selection in cold environments in conjunction with selective polymerization may have been effective in enriching the homochirality of RNA. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b500673b

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Kinetic analysis of hydrolytic reaction of homo- and heterochiral
adenylyl(39–59)adenosine isomers: breaking homochirality reduces
hydrolytic stability of RNA{
Hidehito Urata,* Rie Sasaki, Hiroyo Morita, Marina Kusumoto, Yoko Ogawa, Kozue Mitsuda and
Masao Akagi*
Received (in Cambridge, UK) 14th January 2005, Accepted 8th March 2005
First published as an Advance Article on the web 18th March 2005
DOI: 10.1039/b500673b
The hydrolytic stability of the diastereomeric isomers of ApA
was compared and the results show that heterochiral ApAs are
more rapidly hydrolyzed than homochiral ApAs at low
temperatures, suggesting that hydrolytic selection in cold
environments in conjunction with selective polymerization
may have been effective in enriching the homochirality of
RNA.
There is still no generally accepted explanation for the intriguing
question of the origin of the homochirality of biomolecules.1 The
polymerization of racemic monomers in achiral solution results
in the formation of random polymers composed of D- and
L-repeating units. Therefore, the homochirality of biopolymers
must have been achieved by polymerization after strict chiral
selection of monomers, or more preferably by amplification of a
small chiral imbalance during the formation of biopolymers from
racemic monomers. One possible process for the latter case is the
enantio-selective condensation of racemic or low enantio excess
monomers.2–8 However, the chiral selectivity of such condensation
reactions is not sufficient for producing completely homochiral
polymers. As polymerization often competes with degradation,9–11
the degradation processes of polymers are expected to be another
possible process of enriching the homochirality of polymers.12 In
fact, selective hydrolysis of heterochiral peptides over homochiral
ones has been reported.13 RNA is a polymer that is intrinsically
sensitive to hydrolysis even under very weak alkaline conditions,14
and the selective hydrolysis of heterochiral RNA over homochiral
RNA is predicted,7 based on the expectation of the formation of
less stable secondary and tertiary structures for heterochiral
RNAs. However, such hydrolytic selectivity for heterochiral
RNA oligomers has not yet been reported, and the first attempt
to detect the difference between the hydrolysis rates of the homo-
and heterochiral isomers of uridylyl(39–59)uridine (UpU) under
relatively strong alkaline and acidic conditions was unsuccessful.15
Here, we report a careful kinetic analysis of the hydrolytic
reactions of the diastereomeric isomers of adenylyl(39–59)adenosine
(ApA, Fig. 1) under prebiotically plausible conditions.
Fig. 1 Structures of diastereomeric ApAs.
diminishing rates of the dimers were determined by reversed phase
HPLC. Rate constants for the hydrolysis were obtained by the
first-order rate plots (Fig. S1), and are summarized in Table 1. At
high temperatures (70 and 80 uC), no significant difference in the
rate constant between the homo- and heterochiral isomers was
observed, as reported by Mikkola et al.15 However, a small yet
significant difference in the rate constant was observed at low
temperatures. In fact, the khomochiral/kheterochiral ratios are decreased
as the temperature is decreased (Table 1). It is thus very likely that
the heterochiral ApAs are more easily hydrolyzed than the
homochiral ApAs at low temperatures. The half-lives at 0 uC for
D-(ApA) and L-(ApA) estimated from the Arrhenius plots (Fig. S2)
are 104.9 ¡ 0.9 and 105.4 ¡ 1.7 y, whereas those for ADpAL and
ALpAD are 95.9 ¡ 1.5 and 92.5 ¡ 2.6 y, respectively. Although
the difference in half-life between the homo- and heterochiral
ApAs is small, even such an extent of differential susceptibility of
the oligomers to the hydrolysis may have been effective in
enriching the homochiral RNA oligomers on the time scale of the
Earth. On the other hand, the half-lives at 100 uC of the dimers are
very similar (#1.6 h), therefore, the contribution of the hydrolysis
to the enrichment of the homochiral oligomers would be effective
at low temperatures rather than at high temperatures.
Table 2 shows the activation parameters for the hydrolysis of
ApAs. The values of the activation enthalpy (DH{) of the
homochiral ApAs are slightly larger (#1 kJ mol21) than those
of the heterochiral ApAs, indicating that the strength of the
phosphodiester bond of the homochiral ApAs is intrinsically
higher than that of the heterochiral ones. Besides, the homochiral
dimers showed more positive activation entropies (DS{) compared
with the heterochiral dimers. Therefore, the homochiral ApAs
have lesser constraint in the activation process to the transition
state than the heterochiral ApAs, and thus the preferential
hydrolysis of the heterochiral ApAs at low temperatures is due
to the enthalpic contribution. This means that in the transition
To carefully compare the hydrolytic stabilities of homo- and
heterochiral RNAs, we carried out the hydrolysis of the
diastereomeric isomers of ApA at different temperatures and the
{ Electronic supplementary information (ESI) available: experimental
details and Figures showing the hydrolytic kinetics, the Arrhenius and
Eyring plots. See http://www.rsc.org/suppdata/cc/b5/b500673b/
*urata@gly.oups.ac.jp (Hidehito Urata)
2578 | Chem. Commun., 2005, 2578–2580
This journal is ß The Royal Society of Chemistry 2005

View Article Online
Table 1 Pseudo-first order rate constants for hydrolysis of ApAsa
kobs (s21
Temp.
)
k[D
-(ApA)]
k[A
/
k[L
k[A
D
pA
/
-(ApA)]
(uC)
D-(ApA)
L-(ApA)
ADpAL
ALpAD
L
D
L
]
pA
]
80
70
60
50
40
a
(1.49 ¡ 0.01) 6 1025
(5.00 ¡ 0.02) 6 1026
(1.57 ¡ 0.00) 6 1026
(4.44 ¡ 0.04) 6 1027
(1.12 ¡ 0.01) 6 1027
(1.48 ¡ 0.02) 6 1025
(5.00 ¡ 0.01) 6 1026
(1.56 ¡ 0.01) 6 1026
(4.44 ¡ 0.01) 6 1027
(1.12 ¡ 0.01) 6 1027
(1.50 ¡ 0.01) 6 1025
(5.07 ¡ 0.01) 6 1026
(1.60 ¡ 0.02) 6 1026
(4.58 ¡ 0.09) 6 1027
(1.18 ¡ 0.02) 6 1027
(1.49 ¡ 0.01) 6 1025
(5.07 ¡ 0.04) 6 1026
(1.61 ¡ 0.01) 6 1026
(4.64 ¡ 0.06) 6 1027
(1.19 ¡ 0.01) 6 1027
1.00
0.99
0.98
0.96
0.94
0.99
0.99
0.98
0.97
0.95
Each reaction contained 40 mM dimer, 0.2 M NaCl, 75 mM MgCl2, and 0.1 M HEPES (pH 8.0). Rate constants are means ¡ standard
deviation in triplicate experiments.
the chain terminator,6 and is considered to be a serious hurdle for
the theories of the prebiotic chemical evolution of RNA. The
present results suggest that terminated (heterochiral) oligomers can
be transformed into non-terminated (homochiral) oligomers by the
preferential hydrolysis of terminated oligomers.
Table 2 Activation parameters for hydrolysis of ApAsa
Dimers
Ea (kJ mol21
)
DH{ (kJ mol21
)
DS{ (J mol21 K21
)
112.2 ¡ 0.1
112.2 ¡ 0.2
109.4 ¡ 0.1
109.4 ¡ 0.2
228.74 ¡ 0.26
228.76 ¡ 0.66
D-(ApA)
L-(ApA)
ADpAL
ALpAD
a
111.3 ¡ 0.2
110.9 ¡ 0.3
108.6 ¡ 0.2
108.2 ¡ 0.3
231.06 ¡ 0.58
232.26 ¡ 0.87
In conclusion, we found that the homochiral ApAs are
intrinsically more stable than the heterochiral ApAs under very
weak alkaline conditions. The results suggest that the hydrolytic
instability of RNA and the combined action of polymerization and
hydrolysis during the chemical evolution of RNA may have been
effective in the establishment of the homochirality of RNA.
However, it remains unknown whether RNAs containing other
bases show a similar behavior or not. Studies of the effects of the
other bases and the length of RNA oligomers on the hydrolysis are
currently under way.
Reaction conditions are the same as those in Table 1.
state, the cleavage of the P–O(59) bond of a pentacoordinated
intermediate16 of the heterochiral ApAs is easier than that of the
homochiral ones. The heterochiral ApAs have been shown to have
a more flexible stacked helical conformation than the homochiral
ApAs in solution.17 Therefore, the increase in activation entropy
(DS{) of the homochiral ApAs relative to the heterochiral ApAs
can be explained by the more rigid stacked conformation of the
homochiral ApAs at the ground state.
We thank the Sumitomo Foundation 2003 (Japan) for financial
support.
Two theories for the chemical evolution of biomolecules have
been proposed: one states that the chemical evolution of
biomolecules has proceeded in a hot environment18,19 and the
other postulates the cold origin of life.20 The hot environment,
such as hydrothermal vents, may be more suitable for accelerating
the monomer condensation,18 although it has been reported that
the nonenzymatic polymerization of activated RNA monomers
proceeds effectively even in ice eutectic phases.21 The present
results suggest that the cold environment has a better hydrolytic
selection pressure for homochiral RNA than the hot environment.
It has been predicted that heterochiral polymers are more
susceptible to hydrolytic degradation than homochiral ones, based
on the expectation that heterochiral RNA forms less stable
secondary and tertiary structures.7 Indeed, the duplex stability of
heterochiral RNA is considerably lower than that of homochiral
RNA,22 and double-stranded RNA is more resistant to hydrolysis
than single-stranded RNA.11,23 However, the present results
demonstrate that even in the case of simple single-stranded
oligomers such as ApA, heterochiral oligomers are preferentially
hydrolyzed relative to homochiral ones, and the selective
hydrolysis of heterochiral RNA does not necessarily require the
formation of higher order structures. Although the hydrolytic
selectivity is low, repeated cycles of polymerization and hydrolysis
may have resulted in the enrichment of the homochirality of RNA.
Orgel and coworkers reported the nonenzymatic template-
directed polymerization of activated mononucleotides. This
reaction was strongly inhibited when racemic nucleotides were
employed as the monomer. This phenomenon is called ‘‘enantio-
meric cross-inhibition’’, whereby unnatural L-nucleotides serve as
Hidehito Urata,* Rie Sasaki, Hiroyo Morita, Marina Kusumoto,
Yoko Ogawa, Kozue Mitsuda and Masao Akagi*
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara,
Takatsuki, Osaka, 569-1094, Japan. E-mail: urata@gly.oups.ac.jp;
Fax: +81 (72) 690 1005; Tel: +81 (72) 690 1089
Notes and references
1 J. L. Bada, Nature, 1995, 374, 594–595.
2 A. Saghatelian, Y. Yokobayashi, K. Soltani and M. R. Ghadiri, Nature,
2001, 409, 797–801.
3 H. Zepik, E. Shavit, M. Tang, T. R. Jensen, K. Kjaer, G. Bolbach,
L. Leiserowitz, I. Weissbuch and M. Lahav, Science, 2002, 295,
1266–1269.
4 T. Hitz and P. L. Luisi, Helv. Chim. Acta, 2003, 86, 1423–1434.
5 M. Bolli, R. Micura and A. Eschenmoser, Chem. Biol., 1997, 4,
309–320.
6 G. F. Joyce, G. M. Visser, C. A. A. van Boeckel, J. H. van Boom,
L. E. Orgel and J. van Westrenen, Nature, 1984, 310, 602–604.
7 P. C. Joshi, S. Pitsch and J. P. Ferris, Chem. Commun., 2000, 2497–
2498.
8 H. Urata, C. Aono, N. Ohmoto, Y. Shimamoto, Y. Kobayashi and
M. Akagi, Chem. Lett., 2001, 324–325.
9 G. von Kiedrowski, Nature, 1996, 381, 20–21.
10 G. Wa¨chtersha¨user, Microbiol. Rev., 1988, 452–484.
11 K. Kawamura and M. Umehara, Bull. Chem. Soc. Jpn., 2001, 74,
927–935.
12 Y. Yamagata, H. Sakihama and K. Nakano, Origins Life, 1980, 10,
349–355.
13 N. E. Blair and W. A. Bonner, Origins Life, 1981, 11, 331–335.
14 D. M. Brown, D. I. Magrath, A. H. Neilson and A. R. Todd, Nature,
1956, 177, 1124–1125.
15 S. Mikkola, S. N. Mikhailov, E. Efimtseva, K. Neuvonen, M. Oivanen,
L. Beigelman and H. Loennberg, Origins Life Evol. Biosphere, 2002, 32,
303–310.
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 2578–2580 | 2579

View Article Online
16 R. Breslow and M. Labelle, J. Am. Chem. Soc., 1986, 108, 2655–
2659.
20 J. L. Bada, C. Bigham and S. L. Miller, Proc. Natl. Acad. Sci. USA,
1994, 91, 1248–1250.
17 H. Urata, M. Go, N. Ohmoto, K. Minoura and M. Akagi, Chem.
Commun., 2002, 544–545.
21 P.-A. Monnard, A. Kanavarioti and D. W. Deamer, J. Am. Chem. Soc.,
2003, 125, 13734–13740.
18 E. Imai, H. Honda, K. Hatori, A. Brack and K. Matsuno, Science,
1999, 283, 831–833.
22 H. Urata, H. Shimizu, H. Hiroaki, D. Kohda and M. Akagi, Biochem.
Biophys. Res. Commun., 2003, 309, 79–83.
19 J. A. Baross and S. E. Hoffman, Origins Life, 1985, 15, 327–
345.
23 D. A. Usher and A. H. McHale, Proc. Natl. Acad. Sci. USA, 1976, 73,
1149–1153.
2580 | Chem. Commun., 2005, 2578–2580
This journal is ß The Royal Society of Chemistry 2005