HELICAL STRUCTURES OF HETEROCHIRAL ADENYLYL-(3’-5’)-ADENOSINESAND THEIR ABILITY TO FORM TRIPLE HELIX WITH POLY(U)Hidehito Urata*, Makiko Go, Norihiko Ohmoto and Masao Akagi*
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Email: urata@gly.oups.ac.jp, akagi@gly.oups.ac.jp(Received 22 August 2002 Accepted 30 August 2002)
Abstract
We synthesized four optical isomers -     - of adenylyl-(3’-5’)adenosine (ApA) and investigated the chemical and helical structures of the dimers by means of enzymatic
digestion, circular dichroism (CD) and UV melting experiments. The results of enzymatic digestion experimentswith nuclease P1, snake venom phosphodiesterase (SVPD) and RNase T2 confirmed the chemical structures ofthe dimers. It is known that D-(ApA) and L-(ApA) form right- and left-handed helical structures, respectively [P.
O. P. Ts’o et al. Biochemistry, 9, 3499-3514 (1970)]. The CD spectra of the heterochiral dimers suggested that
ALpAD has a right-handed helical sense whereas ADpAL has a left-handed helical sense. This result was also
confirmed by UV melting experiments of the triple helices formed by the dimers with D-poly(U), which showedthat the thermal stability of D-(ApA)•2poly(U) and ALpAD•2poly(U) is much higher than that of L(ApA)•2poly(U) and ADpAL•2poly(U). Thus, the propensity of ALpAD to form the right-handed helical structure
is similar to that of D-(ApA), whereas L-(ApA) and ADpAL have the similar propensity of resisting the formationof the right-handed helical structure. These results indicate that the chirality of the 3’-end residue is the primaryfactor for determining the helical sense of ApA. On the basis of the above results, the chemical evolution of
RNA and the origin of the homochirality of RNA were discussed.
(Keyword)
homochirality of RNA, adenylyl-(3’of RNA, RNA world5’)-adenosine (ApA), structure of heterochiral RNA, chemical evolution

Viva Origino 30 (2002) 173 - 181© 2002 by SSOEL Japan-173 -

Viva Origino 30 (2002) 173 - 181

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Viva Origino 30 (2002) 173 - 181
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Viva Origino 30 (2002) 173 - 181

Figure 1. Reversed-phase HPLC profiles of D-(ApA) (A), L-(ApA) (B), ALpAD (C) and ADpAL (D) and their reactions with
nuclease P1, RNase T2 and snake venom phosphodiesterase (SVPD). Asterisks represent a peak derived from EDTA. Elution
was carried out on a column of µBondasphere C18-100Å (Waters) with a linear gradient of CH3CN (0-10%) for 20 min in 50
mM KH2PO4, pH 4.0.

Figure 2. Schematic presentation of the results for enzymatic digestion of heterochiral ApAs.

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Figure 3. CD spectra of homochiral ApAs (left) and heterochiral ApAs (right). Concentration of ApAs is 40 µM in 0.1 M
NaCl, 10 mM sodium phosphate, pH 7.0. Measurements were carried out at 0°C.

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Viva Origino 30 (2002) 173 - 181

Figure 4. UV mixing curves of D-(ApA) (A), L-(ApA) (B), ADpAL (C) and ALpAD (D) with poly(U) in 10 mM MgCl2, 10
mM Tris-HCl, pH 7.5 at –5°C. Total nucleotide concentration is 120 µM.

Figure 5. UV melting profiles of the triplexes of D-(ApA)(closed circles), L-(ApA) (open circles), ADpAL (closed
triangles) and ALpAD (open triangles) with poly(U).
Nucleotide concentration is 40 µM for ApAs and 80 µM forpoly(U) in 10 mM MgCl2, 10 mM Tris-HCl, pH 7.5. The
Tm values in the text and Figure 6 were determined fromthe first-derivative plots of the profiles.

Figure 6. Histogram of the melting temperatures of thetriplexes formed by each ApA isomer with poly(U).

-178 -

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Figure 7. Temperature dependence of CD spectra of the triplexes of D-(ApA) (A), L-(ApA) (B), ADpAL (C) and ALpAD (D)with poly(U). Nucleotide concentration is 40 µM for ApAs and 80 µM for poly(U) in 10 mM MgCl2, 10 mM Tris-HCl, pH
7.5. Solid lines represent spectra at –5° and 30°C.

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1.
Gilbert, W., The RNA World, Nature, 319, 618(1986); Gesteland, R. F. and Atkins J. F. Ed.,
The RNA World, Cold Spring Harbor Laboratory
Press, New York, 1993.
2.

Joyce, G. F., Visser, G. M., van Boeckel, C. A.
A., van Boom, J. H., Orgel, L. E., vanWestrenen J., Chiral selection in poly(C)directed synthesis of oligo(G), Nature, 310,
602-604 (1984).

3.

Schmidt, J. G., Nielsen, P. E., Orgel, L. E.,
Enantiomeric cross-inhibition in the synthesisof oligonucleotides on a nonchiral template, J.
Am. Chem. Soc., 119, 1494-1495 (1997).

4.

Ferris, J. P., Ertem, G., Oligomerization ofribonucleotides on Montmorillonite: Reactionof the 5’-phosphorimidazolide of adenosine,
Science, 257, 1387-1389 (1992).

5.

Urata, H., Aono, C., Ohmoto, N., Shimamoto,Y., Kobayashi, Y., Akagi, M., Efficient andHomochiral Selective Oligomerization of
Racemic Ribonucleotides on Mineral Surface,Chem. Lett., 324-325 (2001); Joshi, P. C.,
Pitsch, S., Ferris, J. P., Homochiral selection in
the montmorillonite-catalyzed and uncatalized
prebiotic synthesis of RNA, Chem. Commun.,
2497-2498 (2000).

.

-180 -

Viva Origino 30 (2002) 173 - 181

6.

Tazawa, I., Tazawa, S., Stempel, L. M., Ts'o, P.
O. P., L-Adenylyl-(3’-5’)-L-adenosine and Ladenylyl-(2’-5’)-L-adenosine, Biochemistry, 9,3499-3514 (1970).

7.

Akagi, M., Omae, D., Tamura, Y., Ueda, T.,
Kumashiro, T., Urata, H., A practical synthesisof L-Ribose, Chem. Pharm. Bull., 50, 866-868(2002).

8.

Cimpoia, A. R., Hunter, P. J., Evans, C. A., On
the conversion of arabino- and ribofuranosyl
methyl glycosides to their 1-O-acetyl
derivatives, J. Carbohydr. Chem., 13, 1115-1119(1994).; Vorbrüggen, H., Krolikiewicz, K., New
catalysts for the synthesis of nucleosides,
Angew. Chem., Int. Ed., 14, 421-422 (1975).

9.

Ohtsuka, E., Ohkubo, M., Yamane, A., Ikehara,
M., Studies on transfer ribonucleic acids andrelated compounds. XLIV. A large-scale
synthesis of the anticodon heptanucleotide offormyl-methionine transfer ribonucleic acid byusing 2’-O-tetrahydrofuranylnucleosides, Chem.
Pharm. Bull., 31, 1910-1916 (1983).

10.

/P01, VW, 2345, pp179-191, 67,
1979.

11.

Bush, C. A., Brahms, J., Conformation of
nucleic acids, oligo- and polynucleotides bycirculardichroism
investigations,inPhysicochemical Properties of Nucleic Acids,
Vol. 2, Duchesne, J. ed., chap. 12, Academic
Press, New York, 1973.

12.

Urata, H., Go, M., Ohmoto, N., Minoura, K.,
Akagi, M., Helical structure of heterochiral
RNA dimers: helical sense of ApA is
determined by chirality of 3’-end residue, Chem.
Commun., 544-545 (2002).

13.

Johnson, K. H., Gray, D. M., Sutherland, J. C.,
Vacuum UV CD spectra of homopolymer
duplexes and triplexes containing AüT or AüU
base pairs, Nucleic Acids Res., 19, 2275-2280(1991); Saenger, W., The Principles of Nucleic
Acid Structure, Chapter 10, Springer-Verlag,
New York, 1984.

14.

Inoue, T., Orgel, L. E., Oligomerization of(guanosine
5’-phosphor)-2-methylimidazolide
on poly(C), J. Mol. Biol., 162, 201-217 (1982).

15.

Orgel, L. E., Lohrmann, R., Prebiotic Chemistry
and Nucleic Acid Replication, Acc. Chem. Res.,7, 368-377 (1974).

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