Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (119K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (23)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Sala, M
Right arrow Articles by Wain-Hobson, S
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sala, M
Right arrow Articles by Wain-Hobson, S
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 1996 Oxford University Press 3302-3306

Footnote

Ambiguous base pairing of the purine analogue 1-(2-deoxy- [beta]-D-ribofuranosyl)-imidazole-4-carboxamide during PCR

Ambiguous base pairing of the purine analogue 1-(2-deoxy- [beta]-D-ribofuranosyl)-imidazole-4-carboxamide during PCR Monica Sala , Valérie Pezo , Sylvie Pochet 1 and Simon Wain-Hobson *

Unité de Rétrovirologie Moléculaire and 1 Unité de Chimie Organique, URA CNRS 487, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France Received June 6, 1996; Revised and Accepted July 22, 1996

ABSTRACT

In principle the hydrogen bonding capacities of 1-(2-deoxy- [beta] -d-ribofuranosyl)-imidazole-4-carboxamide (dY), and its N -propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate derivatives (dYTP and dYPrTP) are preferentially incorporated as dATP analogues in a PCR reaction. However, once incorporated into a DNA template their ambiguous hydrogen bonding potential gave rise to misincorporation at frequencies of ~ 3*10-2 per base per amplification. Most of the substitutions were transitions resulting from rotation about the carboxamide bond when part of the template. Between 11-15% of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr behaved in the same way as dY, despite its propyl moiety. These deoxyimidazole derivatives are among the most radical departures from the canonical bases used so far as substrates in PCR and could be used to generate mutant gene libraries.

INTRODUCTION

The frequency of Taq DNA polymerase error generally falls within the range of 2 * 10 -3 to 2 * 10 -4 mutations per nucleotide per amplification ( 1 ). PCR may be rendered highly error prone by using dNTP biases and/or the addition of transition metal cations such as manganese (Mn 2+ ). As a result the mutation rate may be increased to around 10 -1 per base per amplification, while the mutation spectrum can include all four transitions and a sizeable proportion of transversions ( 2 - 4 ). Nucleoside analogues have also been used to decrease the fidelity of PCR. Deoxyinosine (dI) may base pair with dA, dG, dC and dT, although it has a clear preference for dC. Deoxyinosine triphosphate (dITP) could be incorporated by Taq DNA polymerase enhancing the PCR error rate by up to 6-fold and in proportion to the dITP concentration ( 5 ). More recently, the dCTP (6-(2-deoxy-[beta]-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C][1,2] oxazin-7-one triphosphate, dPTP) and dGTP (8-oxo-2'-deoxyguanosine triphosphate, 8-oxodGTP) analogues have been shown to be efficiently incorporated by Taq DNA polymerase ( 6 ). While dPTP yielded predominantly A[middot]G and T[middot]C transitions, the 8-oxodGTP derivative produced mainly A[middot]C and T[middot]G transversions. When combined, very high mutation rates were obtained (best mean of 0.1 per base per amplification) while the proportion of transversions approached 10%.

1-(2-deoxy-[beta]-D-ribofuranosyl)-imidazole-4-carboxamide (dY) is a deoxypurine analogue in which the six-membered ring is opened eliminating C2 and N3 ( 7 ). The carboxamide moiety is free to rotate giving rise to A-like and G-like rotamers (Fig. 1 A). In the A-like conformation dY is analogous to adenosine although an oxygen replaces the nitrogen donor. In such a conformation it may hydrogen bond with T. The G-like conformer mimics guanosine permitting hydrogen bonding with C. Furthermore, if rotation around the glycosidic bond is allowed, dY may base pair with A [A(syn):Y conformation], G [G:Y(syn)] or even itself [Y(syn):Y] (Fig. 1 B). In principle dYTP could be incorporated in a variety of conformations and, once part of the template, could be copied ambiguously. Here it is shown that dYTP, and its propyl derivative dYPrTP (Fig. 1 A), give rise to ambiguous base pairing during PCR.

MATERIALS AND METHODS

1-(2-deoxy-[beta]-D-ribofuranosyl)-imidazole-4-carboxamide (dY) was synthesised as described previously ( 7 ). In the same manner, the 4- N -propylcarboxamide derivative (dYPr) was prepared from the 4- N -propylcarboxamidoimidazole (obtained by reaction of 4- imidazolecarboxylic acid with n -propylamine) via enzymatic transglycosylation using a crude extract of N -deoxyribosyltransferases. The 5'-triphosphate of dYTP and dYPrTP was prepared according to known synthetic procedures. Thus, the 3'-acetylated nucleosides were phosphorylated according to Tener's procedure ( 8 ). The resulting monophosphates were activated as morpholidate ( 9 ) and condensed with tributylammonium pyrophosphate ( 10 ). Both triphosphates were purified by anion exchange chromatography and characterized by mass spectrometry and NMR spectroscopy. The nucleoside triphosphate analogues were taken up at 50 mM in water and frozen. The temperature stability of dYTP and dYPrTP was tested by incubation in PCR buffer at 94oC for 25 min. Analysis by 13 C and 1 H NMR revealed no detectable degradation of the imidazole or carboxamide moieties (data not shown).


Figure 1 . Structure and hydrogen bonding potentials of dY and dYPr. ( A ) In the A-like and G-like conformations, dY is analogous to A and G respectively which may inter-convert by rotation about the carboxamide bond. ( B ) Rotation about glycosidic bonds enables hydrogen bonding with A and G (patterns 3 and 4) and even itself (pattern 5).


Target DNA for PCR was the gene encoding for the type II dihydrofolate reductase (DHFR), encoded by the Escherichia coli plasmid R67 ( 11 ). The amplification primers (3 and 4; 12 ) give rise to a PCR product of 240 bp. PCR reaction conditions were: 10 mM Tris-HCl pH 8.3, 50 mM KCl, 5 mM MgCl 2 , 5 ng plasmid DNA, 100 pmol each primer and 5 U Taq DNA polymerase (Roche-Cetus) in a final volume of 100 [mu]l. dYTP and dYPrTP were added to a final concentration of 1 mM, while dCTP and dTTP were maintained throughout at 200 [mu]M. The dATP and dGTP concentrations varied (Table 1 ). Control PCR reactions were performed under identical conditions yet devoid of an ambiguous base. Thermal cycling parameters were 95oC for 5 min then 50* (95oC, 30 s; 60oC, 30 s; 72oC, 10 min). Long polymerization times were used to favour elongation after dYTP or dYPrTP misincorporation. All reactions were hot started. For temperature dependent amplification the cycling parameters were: 95oC, 5 min; 50* (95oC, 30 s; 35oC or 45oC or 55oC, 10 min).

Table 1 . Mutant frequencies resulting from mutagenic PCR with dYTP or dYPrTP The lower the trim R /ampi R ratio, the greater the degree of hypermutation. Values are good to +- 20%. For all reactions the dTTP and dCTP concentrations were 200 [mu]M. nd, not done.

On the premise that mismatches involving dY or dYPr might be subject to proof-reading post cloning, 10% of PCR products were chased in a second round of PCR using only equimolar canonical dNTPs (200 [mu]M) and 2.5 U Taq DNA polymerase. Thermal cycling parameters were: 95oC, 5 min; 5* (95oC, 30 s; 60oC, 30 s; 72oC, 10 min), 15* (95oC, 30 s; 60oC, 30 s; 72oC, 30 s); followed by 72oC for 10 min. PCR products were purified and cloned as described ( 12 ). Plating out R67 DHFR on trimethoprim (trim) + ampicillin (ampi) plates yields the functional DHFR variants, while plating on ampicillin alone yields the total collection of variants, functional and defective. Consequently the ratio of the former to the latter provides a rapid indication of the efficiency of hypermutagenesis. Given that multiple mutations were sought sequencing was indispensable ( 12 ).

RESULTS

The two products, dYTP and dYPrTP, were first used to completely substitute for one of the four standard dNTPs. The concentrations of the ambiguous novel nucleotide and the three other canonical dNTPs were 200 [mu]M. No PCR product could be discerned in any reaction (data not shown). In an attempt to force incorporation of dYTP and dYPrTP, their concentration was increased to 1 mM and the reaction doped with a low concentration of the dNTP for which dYTP and dYPrTP were substituting. Only when dYTP or dYPrTP were used as deoxypurine analogues could PCR products be recovered. In the case of dYTP (or dYPrTP) as a dATP analogue, [dATP] could be reduced to 2.5 [mu]M (or 5 [mu]M), while as a dGTP analogue, [dGTP] could be reduced to 1 [mu]M (or 1 [mu]M). Below these concentrations no PCR product was recoverable.

When the PCR products were cloned and tested for DHFR activity, the mutagenic effects of the reaction conditions were reflected in trim R /ampi R ratios of less than unity (reactions 1-8, Table 1 ). However, biased dNTP concentrations are themselves mutagenic for PCR necessitating a control without dYTP or dYPrTP in order to ascertain a potential mutagenic effect of the purine analogues (Table 1 , far right). Thus, were the novel nucleotides to be mutagenic, an excess of mutations over the PCR control would be expressed as a lower trim R /ampi R ratio compared to that of the control. From a comparison of these ratios, it was clear that dYTP was mutagenic when used to mimic dATP (reaction 2) or dGTP (reaction 6). By contrast dYPrTP was mutagenic only as a dATP analogue (reaction 3 with respect to reaction 8). No evidence of a significantly altered trim R /ampi R ratio was found when either product was used to mimic both dATP and dGTP at the same time (reactions 9-15).

Table 2 . Mutation frequencies and spectra resulting from dYTP or dYPrTP incorporation during PCR a Reactions are those given in Table 1. b Total number of mutations. c Mutation frequency is the number of mutations scored divided by the product of the number of clones sequences and the target length of the DHFR (231 bp). d Number of transitions (Ti) and transversions (Tv). e The numbers in the heading refer to the hydrogen bonding patterns dY or dYPr could adopt as a substrate or template respectively (Fig. 1B). Thus, a T[middot]A transversion presumably resulted by product incorporation according to pattern 1 followed by copying according to pattern 3.Clones derived from reactions 2, 3 and 6 were sequenced, the relevant data being given in Table 2 . When used to substitute for dATP predominantly T[middot]C and A[middot]G transitions were noted for both products with between 10-15% of T[middot]G,A and A[middot]C,T transversions indicating that dYTP and dYPrTP substrates were accepted essentially in the A-like conformation. A few C[middot]non-C and G[middot]non-G substitutions (~2%) were also noted. The overall mutation frequency was ~3 * 10 -2 per base per amplification and was comparable for the two substrates (Table 2 ). Given that dYTP and dYPrTP behaved essentially as a dATP substrate analogue this means that ~7% of A+T targets could be substituted.

According to the trim R /ampi R ratios, dYTP but not dYPrTP was mutagenic when used in lieu of dGTP (compare reactions 6 and 8, Table 1 ). Sequencing of clones from reaction 6 showed a mutation spectrum dominated by C[middot]T and G[middot]A transitions and a few C[middot]A and G[middot]T transversions (Table 2 ). However, the mutation frequency was some 6-fold lower compared to using dYTP as a dATP analogue. Among the 95 clones sequenced, three insertions/deletions were noted (+1, +3 and -3, among reactions 2, 3 and 6, respectively).

In order to ascertain whether there were any nearest neighbour effects with dY or dYPr induced substitutions, a [chi] 2 analysis of the 5' and 3' base frequencies surrounding the mutations was made. It failed to reveal any significant preference when either dYTP or dYPrTP were used as dATP analogues. However, incorporation of dYTP in lieu of dGTP in the context 5'-CpG-3'/3'-YpC-5' proved significant above that expected ([chi] 2 = 7.0, P <0.01, 1 degree of freedom). No hot spots were identified among the 95 sequences analysed.

In an attempt to increase further the incorporation of ambiguous nucleotides by Taq DNA polymerase, the influence of temperature and manganese cations was investigated. Mutagenic PCR with reaction 3 conditions (Table 1 ) was made at different polymerisation temperatures, notably 35oC, 45oC, 55oC and 72oC. After a chase the PCR products were cloned, transformed and plated on trimethoprim and ampicillin plates. The percent trim R /ampi R ratios showed an inverse correlation with temperature (Fig. 2 ). In other words, although the specific activity of Taq DNA polymerase decreases with temperature ( 13 ) the enzyme is better able to discriminate between canonical dNTPs and their deoxypurine look-alikes. The misincorporation of dNTPs by Taq DNA polymerase can be increased by the addition of manganese cations ( 2 - 4 ). However, addition of 0.5 mM Mn 2+ apparently reduced PCR efficiency so much that no product was recoverable. Control reactions with Mn 2+ yet without the novel substrates readily yielded PCR product suggesting a specific interaction between them and the manganese cations (data not shown).


Figure 2 . Inverse temperature dependence of PCR fidelity (% trim R /ampi R ratio) and PCR polymerization temperature.

DISCUSSION

1-(2-deoxy-[beta]-D-ribofuranosyl)-imidazole-4-carboxamide triphosphate (dYTP) represents a simplified deoxypurine triphosphate analogue resulting from opening the six-membered ring and elimination of C2 and N3 (Fig. 1 A). As a result of rotation about the carboxamide and glycosidic bonds dYTP may, in principle, form hydrogen bonded base pairs with the four canonical bases as well as with itself (Fig. 1 B; 7 ). Although it might be regarded as a highly ambiguous base in view of such properties, the present results suggest that, at least for Taq DNA polymerase, dYTP is only efficiently incorporated as a dATP analogue. dYTP can substitute for dGTP although at a reduced frequency. This may stem from the fact that in solution dYTP predominantly adopts an A-like conformation ( 7 , 14 ). However, once part of a DNA template rotation about the carboxamide bond allows the base to be copied as G or A.

Between 10-15% of substitutions were A[middot]C,T and T[middot]G,A transversions. The A[middot]T and T[middot]A transversions could result from a templated dY template :dA mismatch in the anti:syn conformation (pattern 3, Fig. 1 B). Likewise A[middot]C and T[middot]G transitions probably result from a templated dY template :dG mismatch in the syn:anti conformation (pattern 4, Fig. 1 B). It is interesting to note that the most and least frequent transversions are precisely those the most and least readily accommodated by Taq DNA polymerase ( 15 ). Finally, given a substitution rate of ~3 * 10 -2 per base per amplification, the probability of mutations arising from dY(syn):dY(anti) mismatching (pattern 5, Fig. 1 B) must be considered unlikely.

The mutagenic effect of dYPr needs comment. Rotation about the carboxamide bond generates the G-like rotamer in which the aliphatic propyl side chain points towards the minor groove. Either as a substrate or template such a conformation would be expected to give rise to steric clash. The observation that dYPrTP is not mutagenic as a dGTP analogue, as judged by comparable trim R /ampi R ratios between the reaction and PCR controls (reaction 8, Table 2 ), suggests that it is incorporated even less efficiently than dYTP. However, the comparable substitution frequencies of dYTP and dYPrTP as dATP analogues would suggest that for both, rotation to the G-like conformer is possible when part of the template. Given that Taq DNA polymerase is a monomer, it is possible that different residues contacting the nascent and template strands introduce such asymmetry in the behaviour of dYPrTP as a substrate and dYPr as part of the template.

Substitution frequencies of ~3% obtained with these deoxyimidazole derivatives are comparable to those for a number of other hypermutagenic protocols such as reverse transcription with biased dNTP concentrations ( 2 - 4 ), in vitro T3 transcription using biased NTP concentrations and Mn 2+ cations (V.P. and S.W.-H., unpublished data). Its mutation spectrum is, however, richer than either due to the fact that mutation of both DNA strands occurs. However, the infidelity of PCR with dYTP is currently ~2-3 fold less efficient than hypermutagenic PCR with biased dNTP concentrations plus Mn 2+ cations ( 4 ) or PCR with the modified base dPTP ( 6 ). Nonetheless dYTP has a number of desirable traits: it produces a reasonable proportion of transversions (11-15%), substitutions were apparently random being free of any hot spots or dinucleotide context, while few insertions and deletions were noted. Consequently it could be of use in the construction of mutant gene libraries.

The deoxyimidazole-4-carboxamide triphosphates are possibly among the most radical departures so far from the canonical dNTPs used as substrates in PCR. They give rise to both transitions and a transversions and represent a step towards developing totally ambiguous nucleotides. Further work is directed to elaboration of the heterocycle and/or carboxamide moiety. It would also be interesting to explore just how far the purine moiety may be stripped down and still serve as a substrate for Taq DNA polymerase.

ACKNOWLEDGEMENTS

We would like to thank Laurence Dugué for help with dYTP and dYPrTP synthesis and Philippe Marlière for good discussions. M.S. was supported by the Instituto Superiore della Sanità and Sidaction. V.P. was supported by a bursary from the Ministère pour la Recherche et de la Technologie. The work was supported by grants from Institut Pasteur and l'Agence Nationale pour la Recherche sur le SIDA.

REFERENCES

1 Eckert,K.A. and Kunkel,T.A. (1991) PCR Methods Applic., 1, 17-24.

2 Leung,D., Chen,E. and Goeddel,D. (1989) Technique, 1, 11-15.

3 Fromant,M., Blanquet,S. and Plateau,P. (1995) Anal. Biochem., 224, 347-353.

4 Vartanian,J.P., Henry,M. and Wain-Hobson,S. (1996) Nucleic Acids Res., 24, 2627-2631.

5 Spee,J.H., de Vos,W.M. and Kuipers,O.P. (1993) Nucleic Acids Res., 21, 777-778.

6 Zaccolo,M., Williams,D.M., Brown,D.M. and Gherardi,E. (1996) J. Mol. Biol., 255, 589-603.

7 Pochet,S., Dugué,L., Meier,A. and Marlière,P. (1995) Bioorg. Med. Chem. Lett., 5, 1679-1684.

8 Tenner,G.M. (1961) J. Am. Chem. Soc., 83, 159-168.

9 Moffatt,J.G. and Khorana,H.G. (1961) J. Am. Chem. Soc., 83, 649-663.

10 Moffatt,J.G. (1964) Can. J. Chem., 42, 599-604.

11 Pattishal,K.H., Acar,J., Burchal,J.J., Goldstein,F.W. and Harvey,R.J. (1977) J. Biol. Chem., 252, 2319-2323.

12 Martinez,M.A., Pezo,V., Marlière,P. and Wain-Hobson,S. (1996) EMBO J., 15, 1203-1210.

13 Chen,A., Edgar,D.B. and Trela,J.M. (1976) J. Bacteriol., 127, 1550-1557.

14 Bergstrom,D.E., Zhang,P. and Johnson,W.T. (1996) Nucleosides Nucleotides, 151, 59-68.

15 Huang,M.M., Arnhein,N. and Goodman,M.F. (1992) Nucleic Acids Res., 20, 4567-4573.

Return

* To whom correspondence should be addressed
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
P. A. Kaminski, P. Dacher, L. Dugue, and S. Pochet
In Vivo Reshaping the Catalytic Site of Nucleoside 2'-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution
J. Biol. Chem., July 18, 2008; 283(29): 20053 - 20059.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
H. Strobel, L. Dugue, P. Marliere, and S. Pochet
Synthesis and recognition by DNA polymerases of a reactive nucleoside, 1-(2-deoxy-{beta}-D-erythro-pentofuranosyl)-imidazole-4-hydrazide
Nucleic Acids Res., May 1, 2002; 30(9): 1869 - 1878.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Berger, Y. Wu, A. K. Ogawa, D. L. McMinn, P. G. Schultz, and F. E. Romesberg
Universal bases for hybridization, replication and chain termination
Nucleic Acids Res., August 1, 2000; 28(15): 2911 - 2914.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (119K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (23)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Sala, M
Right arrow Articles by Wain-Hobson, S
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sala, M
Right arrow Articles by Wain-Hobson, S
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?