- Email: [email protected]
Interaction of DNA hairpin loops and a complementary strand by a triplet of base pairs
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 986-99]
December 30, 1988
Interaction of D N A hairpin loops and a c o m p l e m e n t a r y strand by a triplet of base pairs Ulrich B a u m a n n l ) , R o n a l d F r a n k 2) a n d H e l m u t B15cker 2)
1) Max-Planck-In~titut ffzr Biophy~ikalische Chemie, GSttingen, FRG 2) Ge~ell~chaft fi~r Biotechnologische Forschun9, DNA-Synthesegruppe, Braunschweig, FT~G
Received November 7, 1988
Hypothesis of non-enzymatic recognition of primordial t R N A and m R N A precursors is experimentally approached. DNA hairpins containing a different number of deoxyguanosine residues in the loop are analyzed for their binding ability to a chemically fixed single-strand of oligo(dC). In presence of small Mg 2+ concentration a hairpin with five dG residues in the loop is adsorbed to affinity matrix. Comparision of elution t e m p e r a t u r e s of hairpin oligonucleotides with those of single-stranded oligoguanylic acids with length of the loop indicates, that smallest loop able to bind forms a triplet of base pairs. © 1988 Academic
Press,
Inc.
A n i m p o r t a n t p r o b l e m to be overcome during evolution of a t r a n s l a t i o n m a c h i n e r y is w h e t h e r n o n - e n z y m a t i c molecular recognition m i g h t occur exclusively by m e a n s of h y d r o g e n b o n d i n g between c o m p l e m e n t a r y nucleosides. It was d e m o n s t r a t e d t h a t recognition by this m e c h n i s m is at least sufficiently strong a n d specific to allow enzyme-free p o l y m e r i z a t i o n of short oligoguanylates on a p o l y ( C ) - m a t r i x (1,2).
F u r t h e r m o r e , self-replication
of a h e x a d e o x y n u c l e o t i d e w i t h a distinct sequence was r e p o r t e d (3).
By
this possible p r i m o r d i M replication a m u l t i p l i c a t i o n a n d sequence conservation of short R N A - s t r a n d s m i g h t have occured. In order to test a p u t a t i v e
C o r r e s p o n d e n c e to: Ulrich B a u m a n n M a x - P l a n c k - I n s t i t u t ffir Biophysikalische C h e m i e a m Fassberg, D-3400 GSttingen, F R G Abbreviations: hp(dG)3-~, d(ATCCTAG3_sTAGGAT), hp indicating hairpin 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
986
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ribosome-free recognition of simple adaptors acting as primordial tRNA and a mRNA precursor by a triplet of base pairs (4) binding ability of DNA hairpins to a complementary single-strand was recently analyzed. It could be demonstrated that hairpins containing deoxyadenylate residues in its loop bind to oligo(dT)-chains probably by a triplet of base pairs formed between loop nucleosides and the single-stranded chain (5). In order to evaluate if this is a common mechanism independent of the kind of base pair, DNA hairpins with deoxyguanylate residues in the loop were syntheszed and their binding ability to oligo(dC)-cellulose was investigated. The hairpin structure of investigated oligonucleotides adopted at low temperature has been proven by NMR (6) and a recently developed method of accessibility of loop nucleotides to the single-strand-specific nuclease from mung bean (7).
Materials
and
Methods
Materials
Oligonucleotides with hairpin structure 5'-d(ATCCTAG,TAGGAT), n--- 35, were synthesized by the phosphotriester method using segmental solid supports (8). Prior to affinity chromatography last purification step was achieved by preparative polyacrylamide gel electrophoresis (1.5 mm gel). Oligonucleotides p(dG)23,4 were from Pharmacia, Freiburg. Oligo(dC)cellulose (binding capacity of 10-20 A2~0 units poly(I) per g) and oligo(dG)cellulose (binding capacity of 10-20 A~0 units of poly(C) per g) were as well from Pharmacia, Freiburg. Methods Thermostatable glass columns with dimensions 5 x 0.5 cm were used for affinity chromatography of short oligodeoxyguanylates p(dG)2,3,4 as well as hp(dG)3,4,5 on oligo(dC)-cellulose. Binding ability was tested in aqueous solutions with variations of the concentration of Na + and Mg 2+ always in presence of a buffer consisting of 20 mM Tris/HC1, pH 7.2. The method was as described earlier in detail (5), except that before chromatography of p(dG)~ the considered substance was heated for 5' to 80°C in order to disrupt complexes which might have formed by reported self-association of oligoguanylic acids (9, 10). In case of binding to the affinity matrix at 0°C the substance eluted by rise of temperature was analyzed by electrophoresis on a 20 % polyacrylamide gel. Control experiments were performed on an oligo(dG)-cellulose column. 987
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Results and Discussion The studied hairpin oligodeoxynucleotides shown in Fig. 1 have an identical sequence in the double-helical region as those studied recently by NMR (6) and the accessibility of loop nucleotides to single-strand-specific nuclease from mung bean (7).
Therefore, it can be assumed that consid-
ered oligonucleotides adopt a hairpin structure at low temperatures.
As
formation of a d G / d C base pair is accompanied by a bigger free energy, a higher elution temperature can be expected during affinity chromatography as compared to hairpin oligonucleotides containing deoxyadenosine residues in the loop. Hence, salt concentration during affinity chromatography of hp(dG)3,4,5 on oligo(dC)-cellulose was reduced in comparison to analogous experiments performed with deoxyadenosine containing hairpin oligonucleotides. In presence of 20 mM Tris/HC1, pH 7.2, and 0.1 M NaC1 no binding of all of the investigated hairpins occured at 0°C, buffer was not changed in all experiments. In presence of 0.5 M sodium chloride and 5 mM magnesium chloride hp(dG)3 was not bound to oligo(dC)-cellulose, hp(dG)4 was retarded at 0°C and hp(dG)5 was adsorbed to the affinity matrix at 0°C and eluted at 17°C during application of a linear temperature gradient. In presence of 0.1 M NaC1 and 50 mM MgC12 all of the hairpin oligonucleotides were stronger adsorbed to the affinity matrix: hp(dG)~ was slightly retarded, hp(dG)4 was eluted at 18°C and hp(dG)5 was eluted at 38°C. In Fig. 2 chromatographic pattern of the three substances evaluated in presence of 0.5 M NaC1 and 5 mM MgCI~ are shown. Under these conditions the hairpin oligonucleotides containing deoxyguanosine show a very similar behaviour as recently studied hairpins with deoxyadenosine residues in the loop (5). This study revealed that a loop consisting of n nucleotides is able to form n-2 base pairs to the complementary strand. Helix-adjacent loop nucleotides are not oriented to the solution, this could be demonstrated in detail by the method of accessibility of loop nucleotides to the nuclease from mung bean (7). In order to get further evidence supporting this hypothesis short oligoguanylates with the length of the loop were chromatographed under identical conditions. 988
VoI. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c"
¢D o
cq
'6 . o) f:L
I I I I I I
30
0.0
5' d ( A T C C TA~G~
-20 -10
G
3' ( T A G G A T G ~ [n:l 31
Q
Q
E
0
1
2 3 4 5 6 7 E[ution Volume/ml
8
Fig. 1 Primary and secondary structure adopted at lower temperatures of studied oligodeoxynucleotides. Fig. 2 Elution profiles ofhp(dG),~ on oligo(dC)-cellulose. 0.1 A260units in a volume of 30 #l was applied to an oligo(dC)-cellulose column (0.5 x 5 cm) at O°C. Flow rate was 4.6 ml/h; the buffer consists of 20 mM Tris/HC1, pH 7.2; 0.5 M NaC1 and 5 mM MgC12. Arrows indicate start of a considered chromatography. A linear temperature gradient was applied after passage of about 4 column volumes, if the substance was adsorbed to the affinity matrix.
According to the hypothesis hp(dO)4 and p(dG)~ as well as hp(dG)5 and p(dG)3 should reveal a very similar adsorption/elution behavior. In presence of 0.5 M NaC1/5 m M MgC12 M it is indeed observed t h a t hp(dG)~ and p(dG)3 have almost the same elution t e m p e r a t u r e (17 and 12°C respectively). Oligo(dG)2 and h p ( d G ) , show a small deviation as p(dG)2 is not retarded at 0°C, whereas corresponding hairpin oligonucleotide is retarded, this deviation is explained by a known destabilizing effect of 5'-phosphat group of p(dG)~ (11).
All of the dG containing hairpin oligonucleotides
were not adsorbed to oligo(dG)-cellulose in presence of all applied ion concentrations, assay was used as a control to d e m o n s t r a t e base specifity of binding.
At higher Mg 2+ concentration
of 50 raM, as shown
in Table i, the hair-
pin oligonucleotides with n loop nucleotides seem to be adsorbed stronger than oligoguanylates with n-2 residues. This might be caused by additional stabilizing forces due to lateral interactions between helical parts of DNA hairpins. This lateral interactions might be favoured at high Mg 2+ concen989
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1
Adsorption behaviour and elution temperatures of DNA hairpins hp(dG)~ and oligoguanylates p(dG)~ on oligo(dC)-cellulose. Circle (o) indicates a substance not tested, (-) indicates no binding and (ret) indicates retardation at 0°C. Elution temperature in 0.5 M NaCI/5 mM MgCI2 0.1 M NaC1/50 mM MgCI2 hp(dG)~ 2
p(dG)~
hp(dG),
p(dG)~
o
ret
12
ret
21.5
o
3
4
ret
31.5
18
41
5
17
o
38
o
trations leading to the putative formation of an aggregate of closely packed hairpins and a single-strand (4). A similar effect was observed in the analogous series of dA containing hairpins (5). The well known
self-aggregation
of oligoguanylates could also disturb complex formation, although studied hairpins consist only to a minor part of a dG-stretch.
Furthermore,
the
unspecific aggregation due to guanylate clusters can be excluded, as all of the studied hairpins were not adsorbed to oligo(dG)-cellulose.
The effect of
magnesium ions will be studied in further investigations in detail. Nevertheless, the examination of DNA
hairpins at moderate Mg 2+ concen-
tration of 5 mM with different numbers of dG in the loop reveals, that smallest hairpin able to bind is hp(dG)5. It has a similar elution temperature as p(dG)~ indicating the formation of three base pairs by the loop of hairpin oligonucleotide. Same behaviour has been observed in the series of hairpin oligonucleotides containing deoxyadenosine in the loop, whereas dT containing series shows the need of at least a seven-membered loop to allow binding to the complementary chain (5). This is a further support for the model of a primordial translation device presented recently as a triplet of base pairs is postulated to enable primordial non-enzymatic recognition 990
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of adaptor and mRNA precursor (4). The somewhat disturbing effect of higher Mg 2+ concentrations will be investigated in future. Furthermore, efforts will be made to demonstrate a sequence-dependent binding of DNA hairpins containing a distinct Anticodon-like sequence in the loop.
Acknowledgements We wish to thank R. Pohlenz for efforts during preparation of the manuscript. We thank Dr. D. MSbius for support of the work. Part of the work was funded by Bundesministerium ffir Forschung und Technologie. References 1) Fakhrai, H., Inoue, T. and Orgel, L. E. (1984) Tetrahedron 40, 39-45. 2) Joyce, G. F., Visser, G. M., van Boeckel, C. A. A., van Boom, J. H., Orgel, L. E. and van Westrenen, J. (1984) Nature 310, 602-604. 3) yon Kiedrowski, G. (1986) Angew. Chem. 98, 932-935. 4) Kuhn, H. and Waser, J. (1981) Angew. Chem. 93, 495-526. 5) Baumann, U., Lehmann, U., Schwellnus K., van Boom , J. H. and Kuhn, H. (1987) Eur. J. Biochem. 170, 267-272. 6) Haasnoot, C. A. G., de Bruin, S. H., Berendsen, R. G., Janssen, H. G. J. M., Binnendijk, T. J. J., Hilbers, C. W., van der Marel, G. A. and van Boom, J. H. (1983) Y. Biomol. Struct. Dyn. 1, 115-129. 7) Baumann, U., Frank, R. and B15cker, H. (1986) Eur. Y. Biochem. 161, 409-413. 8) Frank, R., Heikens, W., Heisterberg-Moutsis, G. and B15cker, H., (1983) Nucleic Acids Res. 11, 4365-4377. 9) Lipsett, M. N. (1964) J. Biol. Chem. 239, 1250-1255. 10) Lipsett, M. N. (1964) J. Biol. Chem. 239, 1256-1260. 11) Uhlenbeck, O. C., Harrison, R. and Doty, P. (1968) in Molecular associations in biology (Pullman, B., ed.) pp. 107-114, Academic Press, New York. 991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 986-99]
December 30, 1988
Interaction of D N A hairpin loops and a c o m p l e m e n t a r y strand by a triplet of base pairs Ulrich B a u m a n n l ) , R o n a l d F r a n k 2) a n d H e l m u t B15cker 2)
1) Max-Planck-In~titut ffzr Biophy~ikalische Chemie, GSttingen, FRG 2) Ge~ell~chaft fi~r Biotechnologische Forschun9, DNA-Synthesegruppe, Braunschweig, FT~G
Received November 7, 1988
Hypothesis of non-enzymatic recognition of primordial t R N A and m R N A precursors is experimentally approached. DNA hairpins containing a different number of deoxyguanosine residues in the loop are analyzed for their binding ability to a chemically fixed single-strand of oligo(dC). In presence of small Mg 2+ concentration a hairpin with five dG residues in the loop is adsorbed to affinity matrix. Comparision of elution t e m p e r a t u r e s of hairpin oligonucleotides with those of single-stranded oligoguanylic acids with length of the loop indicates, that smallest loop able to bind forms a triplet of base pairs. © 1988 Academic
Press,
Inc.
A n i m p o r t a n t p r o b l e m to be overcome during evolution of a t r a n s l a t i o n m a c h i n e r y is w h e t h e r n o n - e n z y m a t i c molecular recognition m i g h t occur exclusively by m e a n s of h y d r o g e n b o n d i n g between c o m p l e m e n t a r y nucleosides. It was d e m o n s t r a t e d t h a t recognition by this m e c h n i s m is at least sufficiently strong a n d specific to allow enzyme-free p o l y m e r i z a t i o n of short oligoguanylates on a p o l y ( C ) - m a t r i x (1,2).
F u r t h e r m o r e , self-replication
of a h e x a d e o x y n u c l e o t i d e w i t h a distinct sequence was r e p o r t e d (3).
By
this possible p r i m o r d i M replication a m u l t i p l i c a t i o n a n d sequence conservation of short R N A - s t r a n d s m i g h t have occured. In order to test a p u t a t i v e
C o r r e s p o n d e n c e to: Ulrich B a u m a n n M a x - P l a n c k - I n s t i t u t ffir Biophysikalische C h e m i e a m Fassberg, D-3400 GSttingen, F R G Abbreviations: hp(dG)3-~, d(ATCCTAG3_sTAGGAT), hp indicating hairpin 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
986
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ribosome-free recognition of simple adaptors acting as primordial tRNA and a mRNA precursor by a triplet of base pairs (4) binding ability of DNA hairpins to a complementary single-strand was recently analyzed. It could be demonstrated that hairpins containing deoxyadenylate residues in its loop bind to oligo(dT)-chains probably by a triplet of base pairs formed between loop nucleosides and the single-stranded chain (5). In order to evaluate if this is a common mechanism independent of the kind of base pair, DNA hairpins with deoxyguanylate residues in the loop were syntheszed and their binding ability to oligo(dC)-cellulose was investigated. The hairpin structure of investigated oligonucleotides adopted at low temperature has been proven by NMR (6) and a recently developed method of accessibility of loop nucleotides to the single-strand-specific nuclease from mung bean (7).
Materials
and
Methods
Materials
Oligonucleotides with hairpin structure 5'-d(ATCCTAG,TAGGAT), n--- 35, were synthesized by the phosphotriester method using segmental solid supports (8). Prior to affinity chromatography last purification step was achieved by preparative polyacrylamide gel electrophoresis (1.5 mm gel). Oligonucleotides p(dG)23,4 were from Pharmacia, Freiburg. Oligo(dC)cellulose (binding capacity of 10-20 A2~0 units poly(I) per g) and oligo(dG)cellulose (binding capacity of 10-20 A~0 units of poly(C) per g) were as well from Pharmacia, Freiburg. Methods Thermostatable glass columns with dimensions 5 x 0.5 cm were used for affinity chromatography of short oligodeoxyguanylates p(dG)2,3,4 as well as hp(dG)3,4,5 on oligo(dC)-cellulose. Binding ability was tested in aqueous solutions with variations of the concentration of Na + and Mg 2+ always in presence of a buffer consisting of 20 mM Tris/HC1, pH 7.2. The method was as described earlier in detail (5), except that before chromatography of p(dG)~ the considered substance was heated for 5' to 80°C in order to disrupt complexes which might have formed by reported self-association of oligoguanylic acids (9, 10). In case of binding to the affinity matrix at 0°C the substance eluted by rise of temperature was analyzed by electrophoresis on a 20 % polyacrylamide gel. Control experiments were performed on an oligo(dG)-cellulose column. 987
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Results and Discussion The studied hairpin oligodeoxynucleotides shown in Fig. 1 have an identical sequence in the double-helical region as those studied recently by NMR (6) and the accessibility of loop nucleotides to single-strand-specific nuclease from mung bean (7).
Therefore, it can be assumed that consid-
ered oligonucleotides adopt a hairpin structure at low temperatures.
As
formation of a d G / d C base pair is accompanied by a bigger free energy, a higher elution temperature can be expected during affinity chromatography as compared to hairpin oligonucleotides containing deoxyadenosine residues in the loop. Hence, salt concentration during affinity chromatography of hp(dG)3,4,5 on oligo(dC)-cellulose was reduced in comparison to analogous experiments performed with deoxyadenosine containing hairpin oligonucleotides. In presence of 20 mM Tris/HC1, pH 7.2, and 0.1 M NaC1 no binding of all of the investigated hairpins occured at 0°C, buffer was not changed in all experiments. In presence of 0.5 M sodium chloride and 5 mM magnesium chloride hp(dG)3 was not bound to oligo(dC)-cellulose, hp(dG)4 was retarded at 0°C and hp(dG)5 was adsorbed to the affinity matrix at 0°C and eluted at 17°C during application of a linear temperature gradient. In presence of 0.1 M NaC1 and 50 mM MgC12 all of the hairpin oligonucleotides were stronger adsorbed to the affinity matrix: hp(dG)~ was slightly retarded, hp(dG)4 was eluted at 18°C and hp(dG)5 was eluted at 38°C. In Fig. 2 chromatographic pattern of the three substances evaluated in presence of 0.5 M NaC1 and 5 mM MgCI~ are shown. Under these conditions the hairpin oligonucleotides containing deoxyguanosine show a very similar behaviour as recently studied hairpins with deoxyadenosine residues in the loop (5). This study revealed that a loop consisting of n nucleotides is able to form n-2 base pairs to the complementary strand. Helix-adjacent loop nucleotides are not oriented to the solution, this could be demonstrated in detail by the method of accessibility of loop nucleotides to the nuclease from mung bean (7). In order to get further evidence supporting this hypothesis short oligoguanylates with the length of the loop were chromatographed under identical conditions. 988
VoI. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c"
¢D o
cq
'6 . o) f:L
I I I I I I
30
0.0
5' d ( A T C C TA~G~
-20 -10
G
3' ( T A G G A T G ~ [n:l 31
Q
Q
E
0
1
2 3 4 5 6 7 E[ution Volume/ml
8
Fig. 1 Primary and secondary structure adopted at lower temperatures of studied oligodeoxynucleotides. Fig. 2 Elution profiles ofhp(dG),~ on oligo(dC)-cellulose. 0.1 A260units in a volume of 30 #l was applied to an oligo(dC)-cellulose column (0.5 x 5 cm) at O°C. Flow rate was 4.6 ml/h; the buffer consists of 20 mM Tris/HC1, pH 7.2; 0.5 M NaC1 and 5 mM MgC12. Arrows indicate start of a considered chromatography. A linear temperature gradient was applied after passage of about 4 column volumes, if the substance was adsorbed to the affinity matrix.
According to the hypothesis hp(dO)4 and p(dG)~ as well as hp(dG)5 and p(dG)3 should reveal a very similar adsorption/elution behavior. In presence of 0.5 M NaC1/5 m M MgC12 M it is indeed observed t h a t hp(dG)~ and p(dG)3 have almost the same elution t e m p e r a t u r e (17 and 12°C respectively). Oligo(dG)2 and h p ( d G ) , show a small deviation as p(dG)2 is not retarded at 0°C, whereas corresponding hairpin oligonucleotide is retarded, this deviation is explained by a known destabilizing effect of 5'-phosphat group of p(dG)~ (11).
All of the dG containing hairpin oligonucleotides
were not adsorbed to oligo(dG)-cellulose in presence of all applied ion concentrations, assay was used as a control to d e m o n s t r a t e base specifity of binding.
At higher Mg 2+ concentration
of 50 raM, as shown
in Table i, the hair-
pin oligonucleotides with n loop nucleotides seem to be adsorbed stronger than oligoguanylates with n-2 residues. This might be caused by additional stabilizing forces due to lateral interactions between helical parts of DNA hairpins. This lateral interactions might be favoured at high Mg 2+ concen989
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1
Adsorption behaviour and elution temperatures of DNA hairpins hp(dG)~ and oligoguanylates p(dG)~ on oligo(dC)-cellulose. Circle (o) indicates a substance not tested, (-) indicates no binding and (ret) indicates retardation at 0°C. Elution temperature in 0.5 M NaCI/5 mM MgCI2 0.1 M NaC1/50 mM MgCI2 hp(dG)~ 2
p(dG)~
hp(dG),
p(dG)~
o
ret
12
ret
21.5
o
3
4
ret
31.5
18
41
5
17
o
38
o
trations leading to the putative formation of an aggregate of closely packed hairpins and a single-strand (4). A similar effect was observed in the analogous series of dA containing hairpins (5). The well known
self-aggregation
of oligoguanylates could also disturb complex formation, although studied hairpins consist only to a minor part of a dG-stretch.
Furthermore,
the
unspecific aggregation due to guanylate clusters can be excluded, as all of the studied hairpins were not adsorbed to oligo(dG)-cellulose.
The effect of
magnesium ions will be studied in further investigations in detail. Nevertheless, the examination of DNA
hairpins at moderate Mg 2+ concen-
tration of 5 mM with different numbers of dG in the loop reveals, that smallest hairpin able to bind is hp(dG)5. It has a similar elution temperature as p(dG)~ indicating the formation of three base pairs by the loop of hairpin oligonucleotide. Same behaviour has been observed in the series of hairpin oligonucleotides containing deoxyadenosine in the loop, whereas dT containing series shows the need of at least a seven-membered loop to allow binding to the complementary chain (5). This is a further support for the model of a primordial translation device presented recently as a triplet of base pairs is postulated to enable primordial non-enzymatic recognition 990
Vol. 157, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of adaptor and mRNA precursor (4). The somewhat disturbing effect of higher Mg 2+ concentrations will be investigated in future. Furthermore, efforts will be made to demonstrate a sequence-dependent binding of DNA hairpins containing a distinct Anticodon-like sequence in the loop.
Acknowledgements We wish to thank R. Pohlenz for efforts during preparation of the manuscript. We thank Dr. D. MSbius for support of the work. Part of the work was funded by Bundesministerium ffir Forschung und Technologie. References 1) Fakhrai, H., Inoue, T. and Orgel, L. E. (1984) Tetrahedron 40, 39-45. 2) Joyce, G. F., Visser, G. M., van Boeckel, C. A. A., van Boom, J. H., Orgel, L. E. and van Westrenen, J. (1984) Nature 310, 602-604. 3) yon Kiedrowski, G. (1986) Angew. Chem. 98, 932-935. 4) Kuhn, H. and Waser, J. (1981) Angew. Chem. 93, 495-526. 5) Baumann, U., Lehmann, U., Schwellnus K., van Boom , J. H. and Kuhn, H. (1987) Eur. J. Biochem. 170, 267-272. 6) Haasnoot, C. A. G., de Bruin, S. H., Berendsen, R. G., Janssen, H. G. J. M., Binnendijk, T. J. J., Hilbers, C. W., van der Marel, G. A. and van Boom, J. H. (1983) Y. Biomol. Struct. Dyn. 1, 115-129. 7) Baumann, U., Frank, R. and B15cker, H. (1986) Eur. Y. Biochem. 161, 409-413. 8) Frank, R., Heikens, W., Heisterberg-Moutsis, G. and B15cker, H., (1983) Nucleic Acids Res. 11, 4365-4377. 9) Lipsett, M. N. (1964) J. Biol. Chem. 239, 1250-1255. 10) Lipsett, M. N. (1964) J. Biol. Chem. 239, 1256-1260. 11) Uhlenbeck, O. C., Harrison, R. and Doty, P. (1968) in Molecular associations in biology (Pullman, B., ed.) pp. 107-114, Academic Press, New York. 991