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Nonenzymatic Oligomerization of Ribonucleotides on Guanosine-Rich Templates Suppression of the Self-Pairing of Guanosine.

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pear to be generated at intermediate acidities; this phenomenon
requires further investigation.
Given the controversial nature of our findings, we decided to
prepare 4 under our conditions (Scheme 2). Only modest yields
(7.7 %) were obtainable, but this reaction is no doubt complicated by the presence of the third aldehyde grouping. 'HNMR
data again only showed the presence of the expected resonances;
the extra peaks noted by Berlin et al. were absent, even for
spectra recorded at 500 MHz (Figure 1B). The UV/Vis spect r ~ m [ ' for
~ ] the free base gave much higher molar absorptivity
values than reported (the Soret band at 434 nm gave E = 87 600;
E < 40000 was reported by Berlin[']), which is in accord with our
expectations for a porphyrin analog of this type. It is also worth
noting that our samples were obtained as deep purple microneedles and not as the dark green/brown solid previously
isolated.[']
Our new findings demonstrate that no unusual conformationa1 or tautomeric processes are occurring for carbaporphyrins 2
or 4. The anomalous spectroscopic data reported by Berlin
et al.[7.8]are most likely due to impurities arising from the less
favorable reaction conditions used in their studies.[",
Experimental Procedure
2: Dicarboxylic acid 1 [17] (100 mg) was stirred with TFA (1 mL) under an atmosphere of nitrogen for 10 min. CH,CI, (I9 mL) was added followed immediately by
1,3-difonnylindene (38 mg), and the mixture stirred for a further 2 h. The mixture
was neutralized by the dropwise addition of Et,N, DDQ (50 mg) added, and the
resulting solution stirred for an additional 1 h. The mixture was washed with water
and purified by chromatography on neutral Grade 3 alumina with CH,CI, as eluant, and a dark brown fraction was collected. Repeated chromatography on a silica
column with CH,CI, as eluant gave the product a s a broad, brown band. Recrystallization from CHCI,/MeOH afforded 2 (47 mg, 43 %) as fluffy copper/bronze-colored crystals. M.p. = 270 "C (decomp.); UV/Vis (1 % Et,N/CH,CI,): i.,,, (Ig,,s) =
306 (4.32), 376 (4.62), 424 (5.21), 510 (4.25), 544 (4.17), 602 (3.70). 662 nm (3.25);
(IgloE)
,
= 348 (4.60), 426 (5.15), 462 (4.47). 614
UV/Vis (50% TFA/CH,CI,): i,,
(3.85),670nm(4.31);'HNMR(500MHz,CDC1,):6
= - 6.74(s,1 H.H21), -4.0
(v br, 2H, 2 x N H ) , 1.85 (t, 6H). 1.87 (t, 6 H , 4xCH,CH,), 3.64 (s, 6H,
2 x porphyrin CH,). 3.97 (q,4H), 4.07 (q, 4H, 4 x CH,CH,), 7.74 (m. 2H, H2',3').
8.83 (m, 2H, H2',3'), 9.82 (s, 2H, H10,15), 10.10 (s, 2H. H5,20); I3C NMR (75.46
MHz, CDCI,): 6 =11.44, 1740, 18.58, 19.65, 20.05, 95.50, 98.76, 109.68, 120 65,
126.64, 132.50, 133.90, 135.59, 137.83, 141.65, 144.49, 152.87, HR-MS (EI) m/z
calcd for C,,H,,N,: 499.29795; found: 499.29875; elemental analysis calcd for
Selected spectroscopic data for 4: m.p.z300"C; UV/Vis (1 O h Et,N/CH,CI,):
imaX
(Ig,,E) = 316 (4.42), 364 (4.65), 434 (4.94), 524 (4.05), 564 (4.14), 644
(3.48), 706 (3.46); HR-MS (El) m/z calcd for C,,H,,N,O: 477.27801, found.
477.27799.
Fragmentation-recombination processes under acidic conditions can result in
the formation of isomers: A. H. Jackson, W Lertwanawatana, R. K. Pandey,
K. R. N. Rao, J. Chem. Soc. Perkin Truns. I1989, 374.
It should be noted that commercially available CDCI, is often contaminated
with trace amounts of acid, which can result in spectra corresponding to protonated carbaporphyrins. We avoid this problem by purifiying CDC1, over a
short column of basic alumina.
J. L. Sessler, M. R. Johnson, V. Lynch, J. Org. Cl7em. 1987, 52, 4394; T. D.
Lash, J. Porph. Phthal. 1997, in press.
Nonenzymatic Oligomerization of
Ribonucleotides on Guanosine-Rich Templates:
Suppression of the Self-pairing of Guanosine"*
Markus Kurz, Karin Gobel, Christian Hartel, and
Michael W. Gobel*
Dedicated to Professor Gerhard Quinkert
on the occasion of his 70th birthday
The occurrence of information-carrying molecules capable of
propagation and evolutionary development through self-replication, is often seen as a prerequisite for the origin of life.'']
Could RNA have played such an important role? Several results
support this idea. Research carried out primarily by Orgel
et al.
has shown that the oligomerization of 2-methylimidazolides of nucleoside 5'-monophosphates on complementary
oligonucleotide templates can proceed with astonishing efficienThis process, which functions without the help of enzymes, is directed by Watson-Crick base-pairing and by basestacking. Nevertheless, the occurrence of self-replicationr4.51 is
blocked by serious obstacles: The most important of these is
perhaps the tendency of guanosine-rich strands to tetramerize.
This process renders them incapable of serving as templates for
C,,H,,N,~0.25H2O:calcdC83.38,H7.49,N8.33;foundC83.31,H7.29,N8.19.
the synthesis of oIigocytidine units. The second phase of the
replication is thus blocked.[61This problem can be circumvented
Received: October 9, 1996 [Z9649IE]
by the modification of the nucleic acid
for RNA
German version: Angew. Chem. 1997, 109, 868-870
itself, however, the aggregation of guanosine-rich strands has
only been avoided so far by the use of extremely high diluKeywords: aromaticity porphyrinoids . pyrroles
tion.[2b1
Recently we reported the synthesis of an acridine dye that can
[l] T. D. Lash, S . T. Chaney, Angew Chem. 1997,109,687; Angew. Chem. l n t . Ed.
be coupled to nucleic acids by standard methods and that has
Engl. 1997, 109, 1011.
high chemical stability as an advantageous characteristic.['] We
[2] M. J. Broadhurst, R. Grigg, A. W. Johnson, J. Chem. SOC.C 1971, 3681.
have now developed a system for the investigation of template[3] T. D. Lash, Chem. Eur. J. 1996, 2, 1197.
[4] T. D. Lash, Angew. Chem. 1995,107,2703; Angew Chem. l n t . Ed. Engl. 1995,
directed synthesis of RNA oligomers, which is based on the
34, 2533.
extension of acridine-labeled primers. This method offers great
[ 5 ] T. D. Lash, S. T. Chaney, Chem. Eur 1 1996, 2, 944.
flexibility in the experimental setup and allows the equally rapid
[6] T. D. Lash, S. T. Chaney, Tetrahedron Lett. 1996, 37, 8825.
and precise investigation of the kinetics through the use of high[7] K Berlin, C. Steinbeck, E. Breitmaier, Synthesis 1996. 336.
[XI K. Berlin, Angew. Chem. 1996,108,1955; Angew. Chen? I n t . Ed. Engl. 1996,35,
pressure liquid chromatography (HPLC); as we could demon1820.
strate by this technique, the aggregation of guanosine units can
-
191 For example, E. Vogel, W. Haas, B. Knipp, J. Lex. H. Schmickler, Angew
Chent. 1988, 100,445; Angew. Chem. I n f . Ed. Engl. 1988. 27,406
[lo] In ref. [7] any aromatic porphyrinoid with one or more internal CH groups is
considered to be a carbaporphyrin, including tropiporphyrin [6,7]and oxybenziporphyrin [4]. We recommend that the definition of carbaporphyrins be restricted to systems with five-membered carbon rings.
[ l l ] 2. Arnold, Collection Czech. Chem. Comniun. 1965, 30, 2783.
1121 K. Hafner, K H. Vopel. G. Ploss, C. Konig, Lrehigs Ann Chem. 1963,66f, 52.
[13] The recent observation that NH tautomerization for a heptasubstituted "Nconfused" porphyrin, which cdn be considered to be a 2-aza-21-carbaporphyrin, is rapid at room temperature on the ' HNMR timescale, also makes
this claim appear to be somewhat unlikely' B Y . Liu. C. Briickner, D. Dolphin,
Chem. Commun. 1996, 2141
842
8 VCH Verlagsgeselkhqft mbH, 0-69451 Weinhelm, 1997
[*I Prof. Dr. M. W. Gobel, Dip].-Chem. M. Kurz, DipLChem. C. Hartel
Department de Chimie Organique, Universite de Geneve
30 Quai Ernest-Ansermet. CH-1211 Geneve 4 (Switzerland)
Fax: Int. code +(22)328-7396
e-mail: michael.goebel@chiorg.unige.ch
Dr. K. Gobel
Department de Biologie Moleculaire, Universite de Geneve
30 Quai Ernest-Ansermet, CH-1211 Geneve 4 (Switzerland)
[**I This work wassupported by the Fonds der Chemischen Industrie, theDeutsche
Forschungsgemeinschaft, and the Schweizerischer Nationalfonds zur Forderung der wissenschafthchen Forschung.
0570-083319713608-0842$ 17.50-t S0j0
Angew. Chem. Int. Ed. Engl. 1997, 36, No. 8
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be reduced to a minimum by optimization of the reaction conditions.
Our first experiments were carried out with primer 1, which
consists of a 3'-terminal ribo-guanosine and deoxyribonucleotides (Scheme 1 top). Primer 1 forms a stable double helix with
the DNA template 2, whose melting point is approximately
15 K higher than that of the corresponding acridine-free double
helix (Table 1). The single-stranded portion of the complex 1 . 2
serves as the template, which can associate with up to four
Table 1. Thermal stability of the oligonucleotide duplexes tested here
Primer P
c(P) [pM]
c(2) [&MI
r, ["c]
Conditons [a]
1
30
1.2
30
1.2
0.8
0.8
SO
50.2
31 3
35.1
18.8
19.8
19.8
a
a
1
d(GCACG)
d(GCACG)
1
4
2
50
2
0.8
0.8
a
a
b
b
[a] a : 0.25 M TRIS.HC1, pH = 7.65. b: 10 mM phosphate, pH = 7.0, 70 mM NaC1;
detection in each case at 260 nm.
7: R = H
4: R = O H
HO\
rC' 6
OH
HO
dC-dG -T-dG-dC-dC
3dG--f
dG-dC-dA-dC-rG3
OH
HO
OH
-dC-dC -dC
rG*
3
1
complementary guanosine units 3 (Scheme 1 bottom). The
chain extension takes place within the double-helical aggregate
1 -2.(3),. This process can be easily analyzed by reversed-phase
HPLC. While retention times of the usual mono- and oligonucleotides are short, 1 is strongly retained due to its lipophilic
dye component. Chain-extension products of 1 have shorter
retention times with increasing polarity. Fluorescence detection
leads to a further improvement in the detection limit, as shown
by the typical chromatogram in Figure 1a, b. More than 50% of
the primer is converted in only 5 h.[''' The rate of each extension
step can be determined from the integral of the peak (Figure 1 c,
Table 2). It turned out that the last step is as much as 10 to 20
times slower than the two preceding steps. This effect, which has
been observed previously, can be explained by the weak stacking
interactions of the last incorporated mononucleotide.[2a1Surprisingly, also the first step of the extension of 1 is approximately five times slower than the second.
Experiments with the primer 4 have contributed to the understanding of this effect. Primer 4 has an identical sequence to 1,
with the exception that it consists entirely of ribonucleotides.
When 4, as a complex with the template strand 2, is chain-extended by the guanosine unit 3, the first and second extensions
proceed with similarly high rates (Figure 2, Table 2). A comparison of the CD spectra of the primer-template duplexes 1 . 2
and 4.2 helps to explain the difference: Despite identical sequences, the spectra deviate from one another dramatically
(Figure 3). Therefore the duplex conformations must be different. While the spectrum of 1 - 2closely resembles that of a B-helix, the spectrum of 4 . 2 is characteristic for the A-form.["] This
makes the difference in reaction kinetics understandable: The
oligomerization of RNA proceeds optimally only when the
primer- template duplex corresponds to the A-form. Since 1 . 2
is formed compietely from DNA components, it prefers the
B-conformation. The first step is therefore slow. DNA-RNA
hybrids, on the other hand, are fixed in the A-form. When the
first bond is formed between ribonucleotides, the conformation
type of the primer- template duplex alters, with the consequence
that the further coupling steps (with the exception of the last)
proceed more rapidly. The duplex 4 . 2 adopts the A-form from
the beginning. It therefore displays its full reactivity even in the
first step. As a consequence, the overall yield of chain-extended
strands rises on changing to the RNA primer 4, from around 85
to approximately 99 % (of which 90 % are fully extended). This
is of major importance when problematic nucleotides are going
to be incorporated.
A chain extension o f the primers 1 and 4 can be achieved only
when the template 2, the activated building block 3, and Mg2
ions are present. The concentration of N a + is. on the contrary,
insignificant: Oligomerization experiments carried out with 1,2,
and 3 displayed no significant difference in rate in a concentration range from 0.05 to 0.85 M (Table 3). This is important, since
Na+ induces the formation of guanosine tetrads. Self-associa+
Scheme 1. Top. Formulas of the acndine-labeled primers 1 and 4 and the irnidazolides 3 and 6, which were used for chain extension. Bottom: Extension of the
DNA primer 1 on the template 2 by four guanosine units. The black rectangle
denotes the intercalator.
Angeu Chem Int Ed Engl 1997,36. No 8
0 VCH
Verlagsgeselkchaft mbH, 0.69451 Weinheim, 1997
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843
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100
80
0
5
tlh
-
10
15
Figure 2. Percentage product distribution in the extension of the RNA primer 4 on
template 2 as a function of time: B primer (P) 4. o P + 1, 0 P + 2, o P + 3, A P + 4
(approximately 90% after several days).
tlmin
-
n
c) 100
70
60
50
50{
401
40
30
2
20
1
I
-6
10 ° E
20
10
40
30
50
60
70
.
i
l
.
~
240
220
.
.
~
Ilnrn
--
~
280
260
,
~
Figure 1. Analysis of the extension of DNA primer 1 on the template 2. a) UV
peaks and b) fluorescence peaks of a typical chromatogram. From right to left:
Primer, products with one, two, and three additional nucleotides. The mixture of the
unlabeled mono- and oligonucleotides is eluted within the first 5 min and is invisible
to fluorescence detection. c ) Percentage product distribution as a function of time:
m primer (P) 1, o P 1, 0 P + 2, o P + 3, A P + 4.
+
Table 2. Pseudo-first order rate constants (k [h- '1) for chain extension of the DNA
primer 1 and the RNA primer 4 on ternplate 2 (incorporation of the guanosine
building block 3).
k(Primer 1)
0.17
0.96
0.54
0.054
Table 3. Extension of the DNA primer 1(P) on the template 2 (incorporation of the
guanosine building block 3 from P + 1 up to P + 4). Percentage product distribution as a function of the Na+ concentration.
~~~
~
c(Na+) [MI
c(P)
c(P +1)
c(P + 2)
c(P + 3)
c(P + 4)
0.85
0.25
0.15
0.05 [a]
16.8
22.4
24.0
23.2
5.1
5.8
5.6
4.8
9.4
9.7
9.9
8.4
52.9
49.3
48.0
50.6
15.7
12.8
12 4
12.9
[a] Since 3 was used in the form of its Na+ salt, the minimal Na' concentration is
0.05 M .
5
k(Primer 4)
3dGt
1.2
1.5
0.72
0.066
dC-dG-T-dG-dC-dG
-dG-dG-dG'
rG-rC-rA-rC-rG3
rc+
dC-dG-T-dG-dC-dG-dG-dG-dG5'
.
.
.
.
.
.
.
.
.
rG-rc-rA-&-&
tion, which can be monitored by C D spectroscopy, occurs also
with the DNA-template 5 that contains four guanosine units in
sequence (see Scheme 2). While the C D spectrum shows a maximum at 289 nm in Na+-depleted buffers, it shifts to 263 nm in
high Na+ or K + concentrations. Similar changes to the C D
844
,
300
80
tlh-
Step
200
Figure 3. C D spectra of the primer-template duplex from DNA primer 1 and
template 2 (dashed line) and of RNA primer 4 and template 2 (solid line). 2.0 PM
oligonucleotide, 10 mM phosphate (pH =7.0),70 mM NaCI, 5°C. 6 in mdeg
10
0
] ~ Vl
0 VCH VerlagsgesellschaffmbH. 0-69451 Weinheim, I997
+
& -k-rA
.
.
.
.
& rf.$ rc'
.
..
U
-&-rc-rc-rc-rc
3'
Scheme 2. Extension of the RNA primer 4 on the guanosine-rich template 5 by four
cytidine units. The black rectangle denotes the intercalator.
0570-0833/97j3608-0844$ I7.50f .50/0
Angew. Chem. I n f . Ed. Engl. 1997,36, No.8
~
,
~
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spectra are associated with the formation of parallel quadruple
strands." 21
With the complexed primers 1 and 4, the single-stranded portion of template 5 should induce the incorporation of the activated cytidine derivative 6 (Scheme 2). The choice of reaction
conditions, that is the use ofeither a DNA or a RNA primer and
the use of high or low alkali metal ion concentrations, has a
major influence on the outcome of the experiments (Table 4). In
detection at 260 nm; fluorescence: d,, = 355, iem
= 450 nm. The extinction coefficients of the products, which increase with the chain length. were corrected by the
use of empirical factors when the UV peak was integrated.
Table 4. Extension of the primer (P)1 and 4 on the guanosine-rich template 5
(incorporation of the cytidine building block 6 from P + I up to P + 4). In each case
the product distribution is given for a typical experiment.
[I] Reviews: a) G. F. Joyce in Cold Spring Harbor Sjmposiu on Quantitative Biology. Vol. LII, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1987, pp.
41 -51 ; b) A. Kanavarioti, Origins Ltfe Evol Biosphere 1994. 24, 479-494;
c) L. E. Orgel, Arc. Chem. Res. 1995, 28, 109-118.
[2] a)T.Wu, L. E.Orgel,J. Am. Chem. Sot. 1992,114, 317-322;b)ihid. 1992,114,
5496-5501;c)ibid. 1992,114,7961-7969;d)A. R. HilLJr., L. E. Orge1,T. Wu,
Origins Life Evol. Biosphere 1993, 23, 285-290; e) A Kanavarioti, C. F
Bernasconi, D. J. Alberas, E. E. Baird, J. Am. Chem. Soc. 1993, 115, 85378546.
[3] Catalysis of the formation of RNA by metal ions: a) H. Sawai, K. Higa, K.
Kuroda, J. Chem. Soc. Perkin Trans. 1 1992. 505-508; b) R. Rohatgi, D. P.
Bartel, J. W. Szostak, J. Am. Chem. Soc. 1996. 118, 3332 -3339; c) ibid. 1996,
118, 3340-3344; by minerals: d) G. Ertem, J. P Ferris. .Vatwe 1996, 379,
238-240; e) J. P Ferris, A. R. Hill, R. H. Liu, L. E. Orgel, ihid. 1996, 381,
59-61; by peptides: f) B. Barbier, J. Visscher, A. W Schwartz. J. Mol. Evol.
1993,37,554- 558; by ribozymes: g) E. H. Ekland, J. W. Szostak, D. P Bartel,
Science 1995, 269, 373-376; h) E. H Ekland, D P. Bartel. Narure 1996.382,
373-376.
[4) Self-replicating oligonucleotides: a) G. von Kiedrowski, Angriv. Chem. 1986,
98, 932-934; Angen. Chem. Int. Ed. Engl. 1986, 25, 932-935; b) G. von
Kiedrowski, B. Wlotzka, J. Helbing, ibid. 1989, f01. 1259- 1261 and 1989, 28.
1235-1237, c) G. von Kiedrowski, B. Wlotzka, J Helbing. M. Matzen, S.
Jordan, ibid. 1991, 103, 456-459, 1066 and 1991, 30, 423--426, 892; d) D.
Sieves, G. von Kiedrowski, Nature 1994, 36Y, 221 -224; e) T. Li, K. C. Nicolaou, ibid. 1994, 369, 218-221.
[5] Self-replicating, non-nucleic acid based systems: a) A Terfort, G. von
Kiedrowski, Angew. Chem. 1992,104,626-628; Angen. Chrm. hi.Ed Engl.
1992,31,654-656; b) E. A. Wintner, B. Tsao. J. Rebek. Jr., J Org. Chem. 1995,
60, 7997-8001; c) D. N. Reinhoudt, D. M. Rudkevich, F. de Jong, J. Am
Chem. Sot. 1996,118,6880-6889; d) R. Wick, P. Walde, P. L Luisi, ibid. 1995,
117, 1435-1436.
[6] In an experimental approach to find an answer to the question why nature had
preferred RNA and DNA over other plausible alternatives. Eschenmoser and
colleagues investigated "pyranosyl-RNA". In this structure type, the phenomenon of guanosine self-association does not occur, thus making it an excellent substrate for template-directed ligation and replication reactions: S.
Pitsch, R. Krishnamurthy, M. Bolli, S . Wendeborn, A. Holzner. M. Minton, C.
Lesueur, 1. Schlonvogt, B. Jaun, A. Eschenmoser, Helv. Chim. Acta 1995, 78,
1621-1635.
[7] H. Rembold. R. K. Robins, F. Seela, L. E. Orgel, J Mol. Evol 1994, 38, 211 214.
[XI H. Rembold, L. E. Orgel, J. Mol. Evol. 1994, 38, 205-210.
(91 K. Schutz, M. Kurz, M. W. Gobel, Tetrahedron Lert 1995. 36, 8407-8410.
[I 01 Since the products are completely degraded by ribonucleaseT1 t o the phosphorylated primer, in accordance with the finding of Orgel et al the first ribonucleotide must be bound through the 3'-hydroxy group of the primer (see also
Scheme 1 top).
[ I l l D. M. Gray, S.-H. Hung, K. H. Johnson, MerhodsEnzjImo/. 1995,246,19-71.
[12] a) P. Balagurumoorthy, S. K. Brahmachari, D. Mohanty, M Bansal, V. Sasisekharan. N u d Acids Res 1992, 20, 4061 -4067; b) M. Lu, 0. Guo, N. R.
Kallenbach, Biochemistry 1993, 32, 598 - 601
I
[h]
c(P)
c(P + 1 )
c(P + 2 bis
P
DNA-primer 1. I ,M NaCI
4
92.7
6.8
12
82.4
13.2
24
73.3
154
RNA-primer 4. 1 &I KCI
4
89.6
9.2
12
81.4
14.0
18 3
24
75.7
RNA-primer 4, 1 M LiCl
4
51.5
36.7
39 7
12
31 0
24
13.8
25.0
+ 4)
0.5
44
11.3
1.2
4.6
6.0
118
30.4
61 2
c(P)
c(P + 1)
c(P + 2 bis
P 4)
+
DNA-primer I , 50 mM NoCI
81.5
15.6
2.8
58.1
25.1
16.8
45.6
24.6
29.9
RNA-primer 4, 1 M NaCl
75.3
20.2
4.5
56.5
27.2
16.3
42.2
26.5
31.3
RNA-primer 4, SO mM NaCl
52.3
32.7
15.0
27.3
28.4
443
14.8
20.5
64.7 [a]
[a] Approximately 87 % after five days.
accord with earlier results,".'' the extension of DNA primer 1
in 1 M NaCl solution proceeds sluggishly. Lowering the Na'
concentration to 50 mM leads to a strong rate enhancement. The
reactivity of the RNA primer 4 is also reduced by certain alkali
metal ions: very strongly by K + , strongly by N a + , and hardly
at all by Li+ (1 M in each case, Table 4). This effect correlates
with the ability of these ions to induce aggregation of the
guanosine-rich template 5. When high concentrations of Na'
and K ions are avoided, the primer 4 in the system 4.5 does not
react more slowly with cytidine than does, for example, the
primer 1 in the system 1.2 with guanosine.
According to the results described here, the obstacle to replication, caused by self-association of guanosine is not an intrinsic
property of RNA! It appears in fact to be predominantly associated with the high concentrations (more than 1 M) of Na' ions
that have been employed. Under optimized conditions the selfaggregation of short guanosine sequences can be easily avoided
even at template concentrations of 100 PM. We see, thus, much
better chances for the self-replication of RNA through the use
of imidazole-activated mononucleotides than has been thought
possible for a long time.
+
Received: August 19, 1996
Suppiemented version: December 23, 1996 [Z9467IE]
German version. Angew. Chem 1997, 109, 873-876
Keywords: molecular recognition
self-replication
*
oligonucleotides
*
RNA
*
Experimental Section
Oligomerization experiments: Into a 1.5-mL polyethylene tube were pipetted the
following three solutions: buffer, primer 1 or 4, template 2 or 5. After equilibration
for 15 min at room temperature, they were cooled to 10°C. Finally, a freshly prepared solution of 3 or 6 was added, the solutions mixed, the tube sealed, and then
maintained at (1020.2) C. The final concentrations were: 30 p M primer, 100 p M
template, 50 mM imidazolide, 250 mM buffer (TRIS. HCI, pH = 7.7, Na'-free or 1 M
in KCI, NaCl or LiCI; or HEPES, pH =7.7, 1 M NaCI), 200 mM Mg2+.
HPLC analysis: The reaction mixture (3 pL) was diluted with 8 M aqueous urea
solution (10 pL), heated to 90 "C for 1-2 min and then, after cooling, injected onto
the column. Separation conditions: Column Merck LiChrospher 100RP-18 endcapped, 5 pm, 125 x 4 mm. Linear gradient from 5 to 36% A in B in 22 min,
1 mlmin - '. A: 50 mM triethykdmmonium acetate, H,O, pH = 6.5lacetonitrile 401
60, B: 50 mM triethylammonium acetate, H,O, pH = 6.S/acetonitrile 90jlO. UV
Angew. Chem. Int. Ed Engl 1997.36, No. 8
0 VCH
Verlagsgesellschuft mbH, 0-69451 Weinheim, 1997
0570-0833/97/3608-0845 $17.50+ SOjO
845
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nonenzymatic, self, pairing, suppression, oligomerization, ribonucleotide, template, guanosine, rich
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