CYCLIZATION AND DIMERIZATION OF HEXANUCLEOTIDES CONTAINING GUANINE ANDCYTOSINE WITH WATER-SOLUBLE CARBODIIMIDE
Kunio Kawamura* and Fumitaka Okamoto
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University,
Sakai, Osaka 599-8531, Japan
Fax: 0722-54-9903; Email: kawamura@chem.osakafu-u.ac.jp(Received 10 September 2001 Accepted 9 November 2001)(Abstract)
The condensation reaction of hexanucleotides 5'-pGGGCCrC, 5'-pGCGCGrC, and 5'-pGCCCGrG wasinvestigated in the presence of water-soluble carbodiimide (WSC) and imidazole at 0 oC. The efficiency of thecondensation of hexanucleotides with WSC was investigated to know whether these hexanucleotides act as apartial template to promote the elongation reaction. The condensation reaction mainly yielded cyclichexanucleotides (30 - 60 %) and small amount of 12-mers (< 10 %), which opposed our expectation. The yieldof cyclization was in the order 5'-pGGGCCrC ≅ 5'-pGCGCGrC > 5'-pGCCCGrG and that of dimerization was in
the order 5'-pGGGCCrC ≅ 5'-pGCGCGrC < 5'-pGCCCGrG. The trend to cause efficient cyclization could be
an obstacle at an early stage of chemical evolution of RNA.
(Keyword)
Oligonucleotide, RNA, Phosphodiester bond, Cyclicnucleotide, Template-directed reaction, RNA world,
Ligation
Introduction
Ribonucleic acids (RNA) preserve bothinformation and catalytic functions, so the RNA or
RNA-like molecules played important roles under
primitive earth environments [1]. If the RNA world
hypothesis is correct then RNA could have been
accumulated under prebiotic conditions.
The
condensation reactions of nucleotides with andwithout using activated monomer under primitive
earth conditions have been evaluated as primitive
polymerase models of nucleotide oligomers.
Examples include the condensation of activated 5'GMP on a poly(C) template [2], the condensation ofactivated 5'-pApA on a (pU)10 template [3], and thespontaneous condensation of the activated nucleotide
monomer in the presence of metal ions [4] and clay
mineral catalysts [5]. The phosphodiester bondformation using condensation agents have also been
investigated.
Minimal replicating system was
succeeded using short oligonucleotides in thepresence of water-soluble carbodiimide [6].
Moreover, the condensation of hexanucleotide on apoly(U) template [7] and the condensation oftrinucleotide in the presence of trivaline catalyst havebeen evaluated [8].
Based on these prebiotic syntheses ofoligonucleotides, it would be deduced a following
scenario of the chemical evolution of RNA. First, at
the first stage towards the RNA world,
oligonucleotides spontaneously formed in thepresence of catalysts such as metal ions and clay
minerals but without template nucleotides, and thenthe reproduction of ribonucleotides occurred throughpathways such as the template-directed reaction.
Thus, during the spontaneous formation in the

Viva Origino 29 (2001) 162 - 167© 2001 by SSOEL Japan

presence of metal ion and clay catalysts, four types ofbases could have been incorporated into
oligonucleotide sequences.
This may be not
expedient for the replication of oligonucleotides by
the template-directed reaction, since the efficiency of
the template-directed reaction is not high for theformation of pyrimidine oligonucleotides on a purine
polynucleotide template [2]. Thus, it is important tosearch plausible elongation pathways other than theconventional template-directed reaction [3,5]. In
this study, as a connecting pathway between the
spontaneous formation and the replication of RNA,
we supposed that short oligonucleotides could
elongate through a route as shown in Fig. 1a, inwhich the hexanucleotide acts as a partial template ofitself.

Figure 1.
Models of the condensation ofhexanucleotides. (a) double-tracking templatedirected formation of 12-mer from 6-mer. (b)cylization of a hexanucleotide.

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Viva Origino 29 (2001) 162 - 167
To evaluate this model, the condensation ofhexanucleotides with WSC was investigated using
three types of hexanucleotides. Conclusively, thecondensation reaction caused mainly cyclization (Fig.
1b), but the efficiency of the elongation from oligo-61, and -6-2 was very low and that from oligo-6-3 wasslightly high.
Experimental
Hexanucleotides 5'-pGGGCCrC (oligo-6-1), 5'pGCGCGrC (oligo-6-2), and 5'-pGCCCGrG (oligo6-3) were purchased from GENSET (France) as
HPLC purified grade. Ribonuclease T2 (RNase T2)
and alkaline phosphatase (APH) were purchasedfrom SIGMA. All other reagents used were ofanalytical grade. T2
The condensation reactions were performed in an
aqueous solution containing NaCl, MgCl2, imidazole,1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide(WSC), and 0.05 – 0.1 mM hexanucleotide at 0 oC.
The solution containing WSC was freshly preparedfor each reaction. The pH of the sample solutions
was adjusted with 0.1 M NaOH or 0.1 M HCl
solution. The mixture was allowed to stand for 7
days and aliquots were taken over 7 days, which wasimmediately quenched in liquid nitrogen.
Thesamples were analysed by HPLC on a DNA-NPR
anion exchange column from TOSOH Co., Tokyo,
Japan using a gradient of 0 - 1.2 M NaCl at pH 11
with
0.02
M
2-amino-2-hydroxymethyl-1,3propanediol (tris). The kinetic analyses of thereaction curves were performed using the computer
program SIMFIT [6].
The structures of oligonucleotides were
determined by selective enzymatic hydrolyses with
RNase T2 and APH [9]. RNase T2 hydrolysis was
carried out in 0.1 mL reaction mixture for 1 to 18 h at
37 oC at pH 4.5 using 0.05 - 0.5 unit of enzyme, andAPH hydrolysis was performed in 0.1 mL for 30 min
at 37 oC with 1 unit of enzyme.
Results and Discussion
Stability of WSC
It was confirmed by HPLC analysis that both
WSC and imidazole are essential to proceed thecondensation reaction, since oligo-6 in the absence of
WSC or imidazole did not form any products within
7 days. UV spectra of the solution containing NaCl,
MgCl2, imidazole, and WSC was monitored at 200 300 nm for 7 days, but the change in absorbance was
not detected (Fig. 2). Thus, it is regarded that theactivity of WSC remains constant for 7 days at 0 oC
duringthecondensation
experimentsofhexanucleotides. The maximum wavelength of theabsorption spectra of WSC in the absence ofimidazole (213 nm) was sifted to 232 nm in thepresence of imidazole (Fig. 2). Based on these facts,
it is assumed that phosphorimidazolide ofhexanucleotide formed in the presence of WSC andimidazole, and then proceeded the condensation
reactions.

Figure 2. Absorption spectral change of WSC under
the condensation reaction conditions but in theabsence of hexanucleotides.
[WSC] = 0.2 M, [NaCl] = 0.2 M, [MgCl2] =0.075 M, pH = 8.0, 0 oC. Top: 0.1 M imidazole,bottom: no imidazole.
Characterisation of the products
Fig. 1a shows a model that we expected forelongation of hexanucleotides [10]. This model isconsidered as a variation of the template-directedreaction and may be called as the double-track
elongation reaction.
The
3'-terminalofhexanucleotides is RNA and other sequences are
DNA with 5’-phosphate, since it is known that
phosphodiester bond of ribose forms more efficiently
than that of deoxyribose by the condensation reactionin the presence of WSC under several conditions[2,6,8].
In addition,
reasons that thehexanucleotides containing guanine and cytosine
were selected in this study are follows. First,
Watson-Crick base pair formation between guanineand cytosine is stronger than that between adenine
and uracil [11]. Further, it has been repeatedly
described that early genes were constructed from
guanine and cytosine rich nucleic acids rather thanadenine and uracil rich [12].

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Viva Origino 29 (2001) 162 - 167
The reactions of oligo-6-1, -6-2, and -6-3 weremonitored by HPLC and two main products were
found as peak-1 and peak-2 of HPLC (Fig. 3).

Figure 3. HPLC charts of the products from thecondensation of pGGGCCrC (oligo-6-1).
Anion-exchange column (TOSOH Co., DNA-NPR),
HPLC buffer : tris : 0.02 M, NaCl gradient, pH = 11.
The yield of peak-1 was higher than that of peak-2
for three types hexanucleotides tested in this study.
Since the retention time of peak-2 of oligo-6-1 wasconsistent with that of authentic 12-mer (5'pGGGCCCGGGCCC), peak-2 was assigned as a
dimerized product of oligo-6-1.
For further
characterisation of peak-1, enzymatic hydrolysis with
RNase T2 and APH was performed.
Peak-1
disappeared by hydrolysis with RNase T2 so thepeak-1 involves 3’,5’-linked ribose phosphodiester
bond. Besides, peak-1 was not degraded by APH
hydrolysis. Based on these analyses, peak-1 isassigned as cyclic hexanucleotide (cyclic-6-mer) andpeak-2 is assigned as dimerized products (12-mer) of
hexanucleotides. Thus, the condensation reaction
with WSC mainly proceeds cyclization through thepathway as shown in Fig. 1b for oligo-6-1 and -2.
This is coincidence with previous studies, in which
the cyclic nucleotides were easily formed under theprebiotic synthesis of oligonucleotides [4, 9].
The time course of the reactions of oligo-6-1, -2,
and -3 was followed for 24 h at 0 oC (Fig. 4).
Cyclic-6-mers were the main products for oligo-6 anda small amount of 12-mer was observed for oligo-6-1
and -2.
The yield of 12-mer from oligo-6-3 wassomewhat high compared to oligo-6-1 and -2. The
ratio of the extents of elongation to cyclization in
average was 0.045 for oligo-6-1, 0.017 for oligo-6-2,

Figure 4. Reaction profiles for the condensation ofhexanucleotides. [NaCl] = 0.2 M, [MgCl2]= 0.075 M, [Imidazole] =0.1 M, pH = 8.0, 0 oC, [oligo-6-1 or -3] = 1 x 10-4 M,[WSC] = 0.2 M.
(a) 5'-pGGGCCrC, (b) 5'-pGCCCGrG.
and 0.15 for oligo-6-3. The relatively high yield of
12-mer from oligo-6-3 may be due to following
reasons.
First, for the case of oligo-6-1, twomolecules of oligo-6-1 are capable to form double
helix since self-complementary, where the double
helix involves 6 base pairs and does not act as atemplate, and the same type of double helix can beformed for the case of oligo-6-2. However, thepossible double helix involves only 4 base pairs foroligo-6-3 (Fig. 1a). Second, the linkage being
produced by the condensation of oligo-6-1 or -2 is theligation between G and C, but that for oligo-6-3 isthat between G and G. In previous studies, it waselucidated that the formation of associate of activatedmonomer and elongating oligomer is important for
the elongation of oligonucleotides [9,13,14]. The
association between G and G for oligo-6-3 may be
more effective for the formation of 12-mer than thatbetween G and C for oligo-6-1 and –2 (Fig. 2).
Rate of the formation of cyclization and elongation ofhexanucleotides
First, it was observed that salt compositions did
not affect much the condensation reaction. Besides,
the influence of the WSC and oligo-6 concentrations
for the condensation reaction was detected. Boththe yields of cyclic-6-mer and linear 12-mer- 164 -

Viva Origino 29 (2001) 162 - 167
increased in the presence of twice of WSC, while
were not much changed those in the presence of
twice of oligo-6. This is due to that WSC was
added in large excess of hexanucleotide, where thecondensation rate is proportional to the WSC
concentration (Table 1, condition 3,4).
Table 1. The rate constants for the formation of
cyclichexanucleotides and dimerization of hexamers.

1

2

3

4

nulceotides

kcyc / s-1

kdim / s-1 M-1

6-1(4.60.1)10-7

ND

6-2

(6.30.1)10-7

ND

6-3

(2.70.1)10(1.20.2)10-3

6-1(6.40.1)10-7

ND

6-2

(2.90.1)10-7

ND

6-3

(9.91.0)10(6.9.3)10-4

6-1(8.90.3)10-7 (6.02.0)10-4

6-2(6.80.1)10-7 (1.81.1)10-4

6-3(2.80.1)10-7 (8.21.0)10-4

6-1(1.60.1)10-6 (1.20.3)10-3

6-2(1.60.1)10-6 (6.73.9)10-4

6-3(6.90.1)10-7 (1.60.2)10-3-7-8

Reaction Conditions, 1: [NaCl] = 0.1 M, [MgCl2] =0.1 M, [imidazole] = 0.1 M, [WSC] = 0.1 M, pH =7.5; 2: [NaCl] = 1.0 M, [MgCl2] = 0.2 M, [imidazole]= 0.1 M, [WSC] = 0.1 M, pH = 8.0; 3: [NaCl] = 0.2
M, [MgCl2] = 0.075 M, [imidazole] = 0.1 M, [WSC]= 0.1 M, pH = 8.0; 4: [NaCl] = 0.2 M, [MgCl2] =0.075 M, [imidazole] = 0.1 M, [WSC] = 0.2 M, pH =8.0. Hexanucleotides, 6-1: 5'-pGGGCCrC, 6-2: 5'pGCGCGrC, 6-3: 5'-pGCCCGrG.
The reaction curves of oligo-6 (Fig. 4) werefitted using a following reaction model and theapparent rate constants of cyclization (kcyc) anddimerization (kdim) were determined by SIMFIT(Table 1).
oligo-6 cyclic-6-mer(1)oligo-6 + oligo-6  oligo-12(2a)oligo-6 + oligo-6 + oligo-6 oligo-12 + oligo-6 (2b)According to the model shown in Fig. 1a, thedimerzation of oligo-6 should obey pseudo-thirdorder kinetics. However, dimerization was detectedeven in the reactions of oligo-6-1 and –2, which seemto be unlikely to follow pseudo-third order rate.
Thus, both models expressed by equations 2a and 2b
were tested, where pseudo-second-order rate modelgave somewhat better fitting. This fact may indicatethat dimerization of oligo-6s follows pseudo-second-order rate rather than pseudo-third-order rate.
However, since the yield of dimerization is not very
high in these reactions, detail analysis would be
important to determine whether the dimerization is
template dependent or independent. The values ofkdim decreased in the order oligo-6-3 > oligo-6-1 >oligo-6-2.
Besides, the magnitude of kcyc wascomparable for oligo-6-1 and -2, but that of oligo-6-3
was somewhat smaller than that of oligo-6-1 and -2.
The difference between the ligation within G and G
for oligo-6-3 and that within G and C for oligo-6-1
and –2 may be important. These facts suggest thatthe low efficiency of cyclization of oligo-6-3 is also
important factor to determine the yield of
dimerization.
On the Chemical Evolution of RNA
The effective formation of cyclic-6-mer was
unexpected and this is coincidence with someprevious studies, in which rapid cyclization reactions
were
observed
duringtheformationofoligonucleotides from activated nucleotide without
template [4,9]. The cyclization of short peptides
also occurs during the formation of oligopeptides
under prebiotic conditions [15].
Thus, thecyclization of short oligomers during the formation of
biopolymers could have been inevitable underprebiotic conditions since both amino acids andnucleic acids have two functional groups. In other
word, some chemical pathways would be necessary
to prevent from the cyclization of shortoligomolecules. Besides, the dimerization of oligo6 follows pseudo-second or –third-order kinetics sothat the apparent rate of the dimerization is dependent
on the concentration of hexanucleotide, whilecyclization follows pseudo-first-order kinetics.
Thus, dimerization of biomolecules could bepromoted with increasing the concentrations ofmonomer unit.
The yield of oligonucleotides for the
spontaneous formation and the template-directedreaction is dependent on the type of bases, length ofoligonucleotide, and whether 3’,5’- or 2’,5’-linkage.
For example, the spontaneous formations ofoligonucleotides in the presence of metal ion or clay
catalysts are not a base specific reaction [4,5,9], but
the condensation from mixtures of activatedmononucleotides on the clay catalysts is dependent
on the sequence of oligonucleotide and whether
2’,5’- or 3’,5’-linkage [16]. Besides, the templatedirected reaction using an activated mononucleotide
is highly efficient only for the case of the formationof oligo(G) on a poly(C) template [2,17]. Recently,
it was elucidated that the efficiency of the templatedirected reaction can be enhanced by stabilising
double-helical association of elongating unit andtemplate [3]. In these studies on the spontaneous
formation and the template-directed formation ofRNA, the knowledge on the sequence dependence
was limited. The present study showed that the- 165 -

Viva Origino 29 (2001) 162 - 167
elongation and cyclization are possible to besequence dependent and this is useful to estimate
what sequence of oligonucleotides have beeneffectively formed under primitive earth conditions.
Conclusions
In this study, it was found that the condensationof hexanucleotides oligo-6-1, -2, -3 in the presence of
WSC and imidazole yielded mainly cyclichexanucleotides and a small amount of dimerized
product of hexanucleotides.
The fact that theefficiency of cyclization is much higher than that ofdimerization suggests that the cyclization ofoligonucleotides could be a problem for theaccumulation of RNA. Moreover, it was observedthat both the cyclization and dimerization aresequence dependent even though oligo-6-1, -2, and–3 have the same G and C composition.

5.

6.

Acknowledgement
This research was supported by the Sumitomo
Foundation 1998 and the Mazda Foundation's
Research Grant 2000.
We thank Professor T.
Nakahara in Department of Applied Chemistry forthe use of HPLC in Osaka Prefecture University.
Professor G. von Kiedrowski in Ruhr-Universitaet
Bochum generously provided SIMFIT.

7.

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