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Nucleic Acids Research Pages 1271-1274  

An allylic/acyclic adenosine nucleoside triphosphatefor termination of DNA synthesis by DNA template-dependent polymerases
Introduction
Materials And Methods
   Synthesis of 4-adeninyl-2-(hydroxymethyl)-2-butenyl triphosphate (AATP)
   Polymerases and DNA templates
   Polymerase incorporation assays
   Kinetics of incorporation
   Fluorescence sequencing
Results And Discussion
   Synthesis of AATP
   Incorporation assays
   Kinetics of incorporation
   Fluorescence DNA sequencing
Conclusion
Acknowledgements
References

An allylic/acyclic adenosine nucleoside triphosphatefor termination of DNA synthesis by DNA template-dependent polymerases

An allylic/acyclic adenosine nucleoside triphosphatefor termination of DNA synthesis by DNA template-dependent polymerases

Carlos I. Martinez, Lars H. Thoresen, Richard A. Gibbs1 and Kevin Burgess*

Department of Chemistry, PO Box 300012, Texas A & M University, College Station, TX 77842-3012, USA and 1Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA

Received December 14, 1998; Revised and Accepted January 4, 1999

ABSTRACT

An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 µM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq DNA polymerase FS (Taq FS). This result suggests that AATP could be used as a 2[prime],3[prime]-dideoxyadenosine-5[prime]-triphosphate (ddA) surrogate. Kinetics of incorporation revealed that AATP was 48 times less efficiently incorporated than ddA. Furthermore, AATP was used in dye-primer sequencing as a substitute for ddA.

INTRODUCTION

Several terminators of biocatalytic DNA amplification have been described (1-5). These compounds are critical in Sanger-based methods for DNA sequence determination (5). Sanger sequencing relies on 2[prime],3[prime]-dideoxyribonucleoside-5[prime]-triphosphates (ddNTPs) but these are relatively expensive when required in the quantities necessary for large scale sequencing. Cost issues are accentuated in such enormous efforts as the Human Genome Project. Consequently, attempts to obtain easily accessible alternatives are timely (6).

Previously, we reported the syntheses of two acyclic nucleoside triphosphates, N-MeA and N-MeT, and assays of these as surrogates for ddA and ddT, respectively (2). These analogs were similar in structure to peptide nucleic acid (PNA) monomers. It was found that some of the DNA polymerases tested incorporated the analogs with concomitant termination of the polymerization reaction opposite to the corresponding complementary base. However, the concentrations needed for efficient termination were relatively high compared with those of the corresponding dideoxynucleosides.



Mechanistic studies based on the crystal structures obtained for T7 DNA polymerase and Bacillus DNA polymerase I large fragment with a primer-template complex and a nucleoside triphosphate in the active site suggested to us that some conformational restriction was needed for efficient incorporation of triphosphorylated compounds (7,8). In our search for new terminators of DNA polymerases as alternatives for ddNTPs, we studied the possibility of preparing analogs with limited rotational freedom but with a constrained distance between the base and the triphosphate moiety similar to that found within natural nucleoside triphosphates. Herein we present the synthesis of an analog AATP having potential as a ddA surrogate in DNA sequencing.

MATERIALS AND METHODS

Synthesis of 4-adeninyl-2-(hydroxymethyl)-2-butenyl triphosphate (AATP)

9-[4[prime]-Hydroxy-3[prime]-(hydroxymethyl)-2[prime]-butenyl]adenine hydro-chloride (9) (81.5 mg, 0.30 mmol) and 2-chloro-4H-1,3,2-benzodioxaphosrphorin-4-one (54.7 mg, 0.27 mmol) were weighed into a flame-dried 50 ml round bottom flask. Distilled pyridine (0.40 ml) and distilled DMF (2.6 ml) were added under a nitrogen atmosphere. The yellow suspension was stirred for 8 min at 25°C after which tributylamine (0.30 ml, 1.25 mmol) and tributylammonium pyrophosphate (234 mg, 1.26 mmol, solution in 1.5 ml DMF) were added simultaneously. The reaction was then stirred for 15 min and iodine (121 mg, 0.48 mmol) was added as a solution in 12 ml of 98:2 pyridine:water. The reaction was stirred for another 20 min then a 10% (w/v) solution of NaHSO3 was added (~100 drops) to quench the excess iodine. After stirring for another 10 min, the reaction mixture was evaporated to dryness leaving a yellow solid.

The crude solid was dissolved in water and extracted with diethyl ether (2 × 10 ml) and dichloromethane (2 × 10 ml). The aqueous solution was then loaded onto a DEAE-Sephadex A-25 (Sigma) column and eluted with 0-0.5 M TEAB buffer (pH 7) while monitoring the absorbance of the eluant at 260 nm. The fractions containing product (as determined by 31P NMR) were lyophilized to give a white solid. MALDI MS: (M-H-) 455.9 (cyclic triphosphate). The product was further purified by reverse phase HPLC using a Vydac C18 column eluting with a gradient of 5-100% B over 20 min (A = 0.1 M TEAB, pH 7; B = 70% MeCN and 30% A) to give 4.13 µmol (1.4% yield) of the desired triphosphate as a mixture of cis:trans isomers. 31P NMR (121 MHz, 0.1 M EDTA in D2O, H3PO4 external reference) [delta] (p.p.m.) -9.6 (d, J = 24.4 Hz), -10.2 (d, J = 19.4 Hz), -22.0(t, J =20.4 Hz). MS (MALDI): 474 (M-H).

Polymerases and DNA templates

Klenow fragment of DNA polymerase I was purchased from Boehringer Mannheim. Bst DNA polymerase, Moloney murine leukemia virus (M-MuLV) reverse transcriptase and VentR® (exo-) DNA polymerase were purchased from New England BioLabs. rTth DNA polymerase was purchased from Pharmacia. Ampli Taq® DNA polymerase and Ampli Taq® DNA polymerase FS were purchased from Perkin Elmer. Fluorescein-labeled uni-versal primer (5[prime]-TGTAAAACGACGGCCAGT), the running-start oligonucleotide template (5[prime]-TACGGAGGTGGACTGGCCGTCGTTTTACA) and the standing-start oligonucleotide template (5[prime]-TGACTGACTTACTGGCCGTCGTTTTACA) were purchased from Life Technologies (underlined sequence represents that part of the oligonucleotide that remains for amplification). The M13mp19 template was obtained from 10 ml cultures by polyethylene glycol precipitation and purified in 96-well plates using fiberglass filter paper and a protocol developed in our laboratory (10).

Polymerase incorporation assays

Both primer and the running-start template were pre-diluted to a concentration of 1 pmol/µl. An aliquot of 1 µl of each solution was added to the corresponding reaction buffer (for a complete set of optimized conditions for each enzyme see ref. 1) to give a total volume of 5 µl. This mixture was denatured at 80°C for 7 min and allowed to reach room temperature slowly. A mixture containing the corresponding enzyme, dNTPs, ddNTPs or AATP analog (5 µl) was combined with 5 µl of the primer-template complex mixture for a total reaction volume of 10 µl. The reactions were incubated at the optimum temperature for the enzyme being tested for 10 min and stopped by the addition of 5 µl of loading solution (98% deionized formamide, 10 mM EDTA, pH 8.0, 0.025% bromophenol blue, 0.025% xylene cyanol). The stopped reactions were heated to 85°C for 7 min, chilled on ice and 5 µl of each reaction was loaded on a 20% acrylamide gel and electrophoresed at a constant power of 35 W. After electro-phoresis, the gel were transferred to a 10 × 8.25 inch special glass scanning plate and analyzed using a Molecular Dynamics Fluoroimager 595.

Kinetics of incorporation

The primer-template mixture containing the standing-start template was prepared and treated as described in the previous section. Buffer 2× containing 160 mM Tris-HCl (pH 9.0), 4 mM MgCl2 and an appropriate amount (see below) of Ampli Taq DNA polymerase FS were added. The resulting solution was chilled on ice and 10× AATP (10 µM) was added to give a final volume of 10 µl. The amount of polymerase and the reaction time were adjusted so that 1-30% of product was formed linearly with respect to time. Then the enzyme concentration (0.05 U) and the time (6 min) were kept constant and the concentration of the AATP analog (or of ddA) were varied. Analog concentrations ranged from 0.2 to 16 µM and ddA concentrations varied from 0.008 to 0.8 µM (final concentrations). The reactions were quenched using a solution containing 98% deionized formamide, 10 mM EDTA, pH 8.0, 0.025% bromophenol blue, 0.025% xylene cyanol (5 µl) and loaded on 20% acrylamide gels and electrophoresed at 35 W for 2 h. The paramenters of the Michaelis-Menten equation (Vmax and Km) were determined from a non-linear least squares fit to the equation of a rectangular parabola describing the velocity of product formation versus concentration of substrate. Product formation was quantitated using Molecular Dynamics’ fragment analysis software.

Fluorescence sequencing

BODIPY-labeled (Molecular Probes) universal primers, M13mp19 DNA template and Taq FS (0.5 U) were used for the sequencing assays. In control experiments, the dNTPs were used at a final concentration of 80 µM and ddNTPs were used at final concentrations of 0.2 µM for G, C and T and 0.125 µM for the A reaction. The primers were made up to a concentration of 0.1 µM and the M13mp19 concentration was set at 0.1 µg/µl. The dA concentration was varied in those reactions where AATP was tested for incorporation. Reactions containing dA at concentrations of 0, 16 and 80 µM and a final AATP concentration of 30 µM were explored. The final reaction volumes were 7 µl for the C, G and T reactions and 12 µl for the A reactions. They were all carried out in 1× buffer containing 80 mM Tris-HCl and 2 mM MgCl2 at pH 9.0. Primer-template extension experiments were performed using the following thermal cycle method: 95°C/4 s, 55°C/10 s, 70°C/1 min for 14 cycles, then 95°C/4 s, 70°C/1 min for 14 cycles. The combined DNA was precipitated by the addition of cold ethanol (80 µl), centrifuged at 3000 r.p.m. at 4°C, decanted and dried in vacuo. The dried samples were then resuspended in 3 µl of loading dye, heated to 50°C for 5 min, then loaded on a 6% acrylamide gel. Electrophoresis was carried using an ABI Prism[trade] 377 DNA Sequencer.

RESULTS AND DISCUSSION

Synthesis of AATP

Compound 1 was prepared as reported elsewhere. (9) Various attempts were made to selectively protect one of the hydroxyl groups of this diol. Thus attempts were made to form a 2-nitrobenzylidine acetal for a selective ring opening/photo-deprotection approach. However, side reactions complicated formation of the acetal. Reaction of the diol with one equivalent of tBuMe2SiCl gave a mixture of the disilylated products and cis/trans-monosilylated products; the cis/trans-monosilylated materials could be separated from the disilylated product, but not from each other. Some attempts at enzyme-mediated hydrolysis of 1-diacetate were also made, but without success. Finally, we chose to proceed onto the phosphorylation step without deprotection.

Triphosphorylation of this material gave a mixture of cis:trans isomers that we were unable to separate by HPLC, hence it was used directly in the incorporation assays. Triphosphorylation of diol 1 was performed using Eckstein’s procedure (11) as shown in Scheme 1. Initially, the cyclic triphosphate was obtained as observed by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS) (data not shown). However, this intermediate was hydrolyzed to the desired triphosphate under the ion exchange conditions used for purification of the crude material. This was evident in the MALDI-MS of the post-ion exchange fractions where no peak corresponding to the cyclic triphosphate was found whereas a new one was observed for the acyclic triphosphate AATP. AATP was purified via RP-HPLC for the enzymatic experiments.


Scheme 1.

Incorporation assays

Three different DNA templates were used for the incorporation screens. The first assay involved a running-start template with an 11 base 5[prime]-overhang containing only a single T. This T is located two bases away from the 3[prime]-end of the overhang. Six different DNA polymerases were assayed. Of these only rTth DNA polymerase failed to incorporate the acyclic analog. The rest of the enzymes recognized AATP as a ddA surrogate with Taq FS giving the best results (Table 1). In this case a final concentration of only 1 µM of the surrogate was needed for complete termination of the replication reaction (Fig. 2A). This result was unsurprising given that this thermostable enzyme has lower substrate specificity with respect to ddNTPs (12). In our previous studies with N-MeA the minimum effective concentration was 100 µM. Comparison of these two observations implies that the structural features of AATP are advantageous for incorporation.


Table 1. Summary of incorporation of AATP by DNA polymerases

It was not clear in the early stages of this study that termination arose from incorporation of AATP because an alternative mechanism involving misincorporation of a natural dNTP was also possible. An oligonucleotide template with a 5[prime]-overhang containing two contiguous thymidines at the beginning of the sequence was therefore tested to explore this issue. In the presence of only AATP the amplification reaction gave a single base addition product at a 1 µM concentration (Fig. 2B) as with the previous templates. The same template was also used for the determination of Vmax/Km at steady-state kinetics. These observations implied that misincorporation was insignificant in these experiments.

Kinetics of incorporation

Kinetic studies under steady-state conditions were used to determine the efficiency with which Taq FS incorporated AATP compared with ddA. A standing-start primer-template duplex, as described above, was used to study single nucleotide insertion opposite thymidine. The observed Vmax/Km values were 305.9 ± 91.2% µM-1 min-1 and 6.3 ± 1.4% µM-1 min-1 for ddA and AATP, respectively. These values indicate that ddA is incorporated 48 times more efficiently than AATP.


Figure 1. Incorporation of AATP by Taq FS using a running-start template (A) and standing-start template (B). The template sequence is shown to the right of each gel. (A) Lane 1 contained no dNTPs or ddNTPs; lanes 2-7 contained 0.5 µM dCTP. In addition, lane 3 contained 0.1 µM ddA; lane 4 contained 0.5 µM dATP and 0.5 µM dT. Lanes 5-7 contained 0.1, 1.0 and 10 µM AATP, respectively. (B) Lane 1 contained no dNTPs or ddNTPs; lane 2 contained 0.5 µM dA; lanes 3-5 contained 0.05, 0.5 and 5.0 µM ddA, respectively; lanes 6-10 contained 0.1, 1.0, 10 and 100 µM and 1 mM, respectively.


Figure 2. Electropherogram sections for the sequence analysis of a M13mp19 template with BODIPY-labeled primers. (A) Sequence analysis using 80 µM dNTPs, 0.2 µM ddNTPs (ddC, ddG and ddT) and 0.125 µM ddA. (B) Sequence analysis using the same conditions as above but using 0.125 µM AATP instead of ddATP. (C) Sequence analysis using 80 µM dNTPs, 0.2 µM ddNTPs (ddC, ddG and ddT) and 30 µM AATP.

Fluorescence DNA sequencing

Dye-primer fluorescence DNA sequencing was used to further investigate AATP’s potential as a ddA surrogate. BODIPY-labeled oligonucleotides were used as primers (13) and a M13mp19 DNA used as the template. Initially, the reactions were conducted using AATP as a ddA substitute at the same optimized final concentration established for the latter (0.125 µM). However, the intensities of the termination signals were not even throughout the electropherogram. Indeed, for poly(CA) regions (i.e. CACAC) the A signals were very weak or were not observed at all (Fig. 2B). Poly(A) regions of M13, however, had stronger intensities sometimes going off-scale. This problem suggested that the ratio of AATP to dA had to be adjusted to read over these difficult sequence regions.

In a different experiment the concentration of dA was varied from 0 to 80 µ[Mgr] while keeping that of AATP constant at 30 µM. Figure 2C shows that when the concentration of dA was 80 µ[Mgr], the electropherogram was significantly improved. Signals corres-ponding to poly(A) regions were more even and peak intensities for the poly(CA) region were increased such that both signal quality and base calling efficiency were drastically improved. We are currently investigating other ratios and conditions to further optimize the signal amplitude regularity for the A reactions.

CONCLUSION

An allylic/acyclic adenine triphosphate (AATP) has been inves-tigated as a terminator of biocatalytic DNA replication. AATP was obtained as an inseparable mixture of cis:trans isomers. This mixture proved to be an excellent ddA surrogate when used in conjunction with several DNA polymerases. Assays using a short running-start template showed termination by incorporation opposite T with all but one of the polymerases used. Taq FS, however, gave the best results requiring only a 1 µM concentration of AATP for complete termination. Standing-start incorporation experiments were used to rule out the possibility of competing dNTP misincorporation. Steady-state kinetics for the single base insertion of either ddA or AATP were used to compare the efficiency of incorporation of these analogs by Taq FS. Values of Vmax/Km showed that ddA was only 48 times better than AATP as substrate. This was in accord with the qualitative observations from the standing-start assay. Fluorescence DNA sequencing was then used to further test AATP’s feasibility for high throughput DNA sequencing. The results obtained after testing different ratios of dA:AATP clearly showed that 80:30 µM gave regular incorporation at the expected positions.

It is likely that one of the isomers in the cis/trans mixture of AATP was incorporated more efficiently than the other. The fact that the cis:trans ratio was ~1:1 implies that the concentration of AATP used in the incorporation experiments described here was up to twice that actually required if a pure analog was used (this assumes that the less active analog does not inhibit the polymerase). Unfortunately, the cis and trans isomers of AATP are difficult to separate or prepare stereochemically pure. However, analogs of AATP containing only one alcohol functionality warrant further study and this area should be pursued.

ACKNOWLEDGEMENTS

We would like to thank Dr Steve Scherer for helpful discussions, Linda Savage for assistance with fluorescence sequencing experiments, Dr Hiram Gilbert for guidance with kinetic experiments and Mr Alex J. Zhang for the MALDI-MS analyses. Financial support was provided by the Texas Advance Technology Program and The Robert A. Welch Foundation, and NIH grant HG01745. K.B. thanks the NIH for a Research Career Development Award and the Alfred P. Sloan Foundation for a fellowship. C.M. thanks MBRS-NIH for a pre-doctoral fellowship.

REFERENCES

1. Metzker,M.L., Raghavachari,R., Richards,S., Jacutin,S.E., Civitello,A., Burgess,K. and Gibbs,R.A. (1994) Nucleic Acids Res., 22, 4259-4267. MEDLINE Abstract

2. Martinez,C.I., Ansari,M.A., Gibbs,R. and Burgess,K. (1997) Bioorg. Med. Chem. Lett., 7, 3013-3016.

3. Chidgeavadze,Z.G., Beabealashvilli,R.S., Krayevsky,A.A. and Kukhanova,M.K. (1986) Biochim. Biophys. Acta, 868, 145. MEDLINE Abstract

4. Cocuzza,A.J. (1988) Tetrahedron Lett., 29, 4061-4064.

5. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl Acad. Sci. USA, 74, 5463-5467. MEDLINE Abstract

6. Hobbs,F.W. and Cocuzza,A.J. (1988) European patent 87305844.0.

7. Kiefer,J.R., Mao,C., Braman,J.C. and Beese,L.S. (1998) Nature, 391, 304-307. MEDLINE Abstract

8. Doublié,S., Tabor,S., Long,A.M., Richardson,C.C. and Ellenberger,T. (1998) Nature, 391, 251-258. MEDLINE Abstract

9. Haines,D.R., Tseng,C.K.H. and Marquez,V.E. (1987) J. Med. Chem., 30, 943-947. MEDLINE Abstract

10. Anderson,B., Lu,J., Edwards,K.E., Muzny,D.M. and Gibbs,R.A. (1996) Biotechniques, 20, 1022-1027. MEDLINE Abstract

11. Ludwig,J. and Eckstein,F. (1989) J. Org. Chem., 54, 631-635.

12. Tabor,S. and Richardson,C.C. (1995) Proc. Natl Acad. Sci. USA, 92, 6339-6343. MEDLINE Abstract

13. Metzker,M.L., Lu,J. and Gibbs,R.A. (1996) Science, 271, 1420-1422. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 409 845 4345; Fax: +1 409 845 8839; Email: burgess@mail.chem.tamu.edu

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