The analogue of thymidine triphosphate containing a methylene group in place of the 5′ oxygen can serve as a substrate for reverse transcriptase

Article abstract of DOI:10.1081/NCN-120022031

The thymidine 5′-triphosphate analogue containing a methylene group in place of the 5′ oxygen atom can be prepared using modifications of published procedures and can substitute for the natural thymidine triphosphate in chain extension reactions catalyzed

Full text of DOI:10.1081/NCN-120022031

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
The Analogue of Thymidine Triphosphate
Containing a Methylene Group in Place of the
5′ Oxygen Can Serve as a Substrate for Reverse
Transcriptase
M. A. Bell , G. R. Gough & P. T. Gilham
To cite this article: M. A. Bell , G. R. Gough & P. T. Gilham (2003) The Analogue of Thymidine
Triphosphate Containing a Methylene Group in Place of the 5′ Oxygen Can Serve as a Substrate
for Reverse Transcriptase, Nucleosides, Nucleotides and Nucleic Acids, 22:4, 405-417, DOI:
10.1081/NCN-120022031
To link to this article: http://dx.doi.org/10.1081/NCN-120022031
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Date: 06 November 2015, At: 06:48

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 4, pp. 405–417, 2003
The Analogue of Thymidine Triphosphate Containing a
Methylene Group in Place of the 50 Oxygen Can Serve
as a Substrate for Reverse Transcriptase
*
M. A. Bell, G. R. Gough, and P. T. Gilham
Department of Biological Sciences, Purdue University, West Lafayette,
Indiana, USA
ABSTRACT
The thymidine 50-triphosphate analogue containing a methylene group in place of
the 50 oxygen atom can be prepared using modifications of published procedures
and can substitute for the natural thymidine triphosphate in chain extension reac-
tions catalyzed by Moloney-MLV reverse transcriptase. Using rabbit b-globin
mRNA as the template together with an appropriate primer, the enzyme readily
makes full-length DNA transcripts in which all thymidine 50 oxygen atoms have
been replaced with methylene groups. In sequence analyses using the partial
depurination procedure, the analogue DNA transcript produces electrophoretic
gel patterns identical with those of the corresponding natural DNA transcript.
Experiments on second strand synthesis using the four regular triphosphates show
that the analogue DNA transcript, like the natural transcript, can serve as a tem-
plate. The two DNA duplexes (natural=natural and analogue=natural) formed by
these reactions produce similar electrophoretic cleavage patterns when treated
with either of the endonucleases HaeIII and EcoRI. However, further studies
on template properties indicate that, while the enzyme makes a full-length pro-
duct when using the analogue substrate with a natural DNA strand as template,
it appears unable to use the analogue transcript as template with the analogue
*Correspondence: P. T. Gilham, Department of Biological Sciences, Purdue University,
West Lafayette, IN 47907, USA; E-mail: pgilham@purdue.edu.
405
DOI: 10.1081/NCN-120022031
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

406
Bell, Gough, and Gilham
triphosphate as substrate during second strand synthesis. Preliminary experiments
have also been carried out with a DNA polymerase. No products are detected in
reactions using Taq polymerase with PCR protocols containing the analogue tri-
phosphate as the only source of thymidine.
Key Words: Reverse transcriptase; Phosphonate; Thymidine triphosphate;
Nucleotide analogue.
INTRODUCTION
Studies on nucleic acid molecules containing specific chemical modifications can
yield new insights into their structures and functions and suggest new directions for
drug design in the therapeutic control of human diseases. The modification of parti-
cular interest in this laboratory has been the substitution of a methylene group for
the 50 oxygen within the nucleic acid backbone structure. The initial observations
were made in work on RNA synthesis, where it was shown that the adenosine 50-
diphosphate analogue containing the methylene substitution for the 50 oxygen could
be readily polymerized by polynucleotide phosphorylase.[1,2] The oligonucleotide
products of this reaction are apparently capable of assuming conformations that
are essentially isosteric with their natural counterparts since they were shown to form
triple-stranded complexes with uridine oligomers that are analogous to those formed
from adenosine oligomers containing the natural phosphodiester linkage. Extension
of this work indicated that the corresponding analogue of adenosine 50-triphosphate
can substitute for ATP as a substrate for T3 RNA polymerase.[3] We now show that
the similar analogue of the deoxyribonucleotide thymidine 50-triphosphate is an
acceptable substrate for M-MLV reverse transcriptase.
MATERIALS AND METHODS
Materials
Nucleosides, 1,3-dicyclohexylcarbodiimide, 1,10-carbonyldiimidazole, and pyri-
dinium trifluoroacetate were obtained from either Sigma Chemical Co. or Aldrich
Chemical Co. Chloromethylphosphonic dichloride was obtained from Alpha Pro-
ducts, and converted to diphenyl chloromethylphosphonate using a published proce-
dure.[4] Pyridine was purified by distillation and then maintained in an anhydrous
state by storage over calcium hydride. Dimethylformamide was dried over 4A mole-
cular sieves. Silica gel 60 and aluminum-backed silica gel 60 F254 TLC plates were
obtained from EM science. 30-O-Acetylthymidine was prepared as described in the
literature.[5] The Wittig reagent diphenyl (triphenylphosphoranylidene)methyl-
phosphonate was prepared by condensing diphenyl chloromethylphosphonate with
triphenylphosphine using a published procedure.[6] Crotalus Adamanteus venom
phosphodiesterase I, NAP columns containing DNA grade G-25 Sephadex, and
ultrapure deoxyribonucleoside 50-triphosphates were purchased from Pharmacia
Biotech Inc. Rabbit b-globin mRNA and Moloney-MLV reverse transcriptase

dTTP Analogue
407
(RNase Hꢀ, Superscript II) were obtained from Gibco BRL Products. Ribonuclease
inhibitor and Microcon microconcentrators were purchased from Promega and
Amicon, respectively. Oligodeoxyribonucleotide primers were synthesized by Inte-
grated DNA Technologies, Inc., and labeled at their 50 ends with radioactive phos-
phate using phage T4 polynucleotide kinase and [g-32P]ATP.
Chromatography
HPLC was performed on a Varian 5000 Liquid Chromatograph utilizing either a
Supelcosil LC-SI silica gel column (0.5 cm ꢁ 15 cm) from Supelco that had been deri-
vatized with polyethyleneimine (PEI) prior to use[7] or a Varian Micropak SAX-10
column (0.4 cm ꢁ 30 cm). The solvent system used for elution of products from the
Supelcosil PEI column was aqueous 0.05 M KH2PO4 containing 30% (v=v) methanol
and a linear gradient of 0–0.2 M (NH4)2SO4 at pH 6 with a flow rate of 1.0 mL=min.
The solvent system used with the Varian SAX-10 column was 0.025 M KH2PO4
containing 30% (v=v) methanol with a flow rate of 1.0 mL=min. Silica gel chromato-
graphy was carried out on columns (2.5 cm ꢁ 30 cm) of silica gel 60 packed in a
chloroform slurry, and elution of products was achieved with various concentrations
of methanol in chloroform. Thin layer chromatography used aluminum-backed
silica gel 60 F254 plates (4 cm ꢁ 7 cm), and development was carried out in glass
chambers using various concentrations of methanol in chloroform. Preparative
paper chromatography was conducted in the descending mode on Whatman
3 MM paper, using solvent systems: A, isopropanol=conc. NH4OH=water (7:1:2,
v=v=v); B, isobutyric acid=1 M aqueous NH4OH (10:6, v=v); or C, n-propanol=conc.
NH4OH=water (55:10:35, v=v=v).
Electrophoresis
Denaturing polyacrylamide gel electrophoresis was performed using 11% or 15%
acrylamide cross-linked with N,N0-ethylenebisacrylamide and cast in Tris borate-
EDTA buffer containing 7 M urea. Gel polymerization was initiated by the addition
of 10% (w=v) ammonium persulfate and N,N,N0,N0-tetramethylethylenediamine.
Diphenyl ester of 30-O-acetyl-50-phosphonomethyl-50-deoxythymidine. 30-O-
Acetylthymidine (427 mg, 1.5 mmol) was dissolved in 4.1 mL of anhydrous dimethyl
sulfoxide. To this solution were added with stirring dicyclohexylcarbodiimide
(929 mg, 4.5 mmol), pyridinium trifluoroacetate (145 mg, 0.75 mmol), and pyridine
(61.0 mL, 0.75 mmol). The solution was sealed under argon and stirring was contin-
ued overnight at room temperature. The resulting 50-aldehyde was treated directly
with diphenyl (triphenylphosphoranylidene)methylphosphonate (763 mg, 1.5 mmol),
and the mixture was stirred for a further two days at 37ꢂC under argon. It was then
cooled to room temperature and dissolved in 150 mL ethyl acetate. The solution was
extracted with 10% NaCl, then dried over sodium sulfate and evaporated to an oil
under reduced pressure. Silica gel chromatography with CHCl3 containing a step-
wise gradient (0–2.5%) of MeOH yielded the diphenyl ester of 50,60-dehydro-30-O-
acetyl-50-phosphonomethyl-50-deoxythymidine in 89% yield. The ester (266 mg,

408
Bell, Gough, and Gilham
0.5 mmol) was dissolved in 13.2 mL ethanol and added to 10% palladium on acti-
vated carbon (115 mg). This mixture was then stirred under H2 for 3 days at room
temperature. The reaction gave the reduced diphenyl ester of 30-O-acetyl-50-phos-
phonomethyl-50-deoxythymidine in quantitative yield as demonstrated by 31P-
NMR and TLC.
50-Phosphonomethyl-50-deoxythymidine. The monophenyl ester of 50-phosphono-
methyl-50-deoxythymidine was produced by dissolving the diphenyl ester (90 mg,
0.175 mmol) in a mixture of 1.0 mL 1,4-dioxane and 1.0 mL 1 M NaOH. After 2 h
at room temperature, the solution was neutralized with Dowex 50W-X2 (Hþ form)
ion-exchange resin until a pH of ca. 7 was indicated. The solution was then filtered
and evaporated under reduced pressure to a minimal volume. The products were
fractionated by paper chromatography using solvent system A, affording the pure
monophenyl ester in 95% yield. The monophenyl ester (1,315 AU260, 0.137 mmol)
was then incubated in a mixture of 2.5 mL of 100 mM ammonium acetate (pH 8.9)
containing 14 mM MgCl2 and 10 mL of snake venom phosphodiesterase I
(56 units=mL). The hydrolysis reaction was allowed to proceed for 3 days at room
temperature with daily additions of 5 mL of phosphodiesterase I. The reaction mix-
ture was co-evaporated (3 ꢁ 5 mL) with water, and the resulting oil dissolved in a
minimal volume of water and purified by paper chromatography using solvent
system C. Elution from the paper gave the ammonium salt of 50-phosphono-
methyl-50-deoxythymidine in 83% yield; its purity was established by chromato-
graphy on the Micropak SAX-10 column.
50-Phosphonomethyl-50-deoxythymidine diphosphate. Sodium pyrophosphate
decahydrate (2.23 g, 5.0 mmol) was converted to its pyridinium form by passage
through a column of Dowex 50W-X2 (pyridinium form) ion-exchange resin using
5% aqueous pyridine as eluant. The product was concentrated in vacuo and added
to tri-n-butylamine (2.4 mL, 10 mmol). The mixture was dried by repeated co-
evaporation with anhydrous pyridine (4 ꢁ 10 mL), and then with anhydrous DMF
(2 ꢁ 10 mL). The product was finally brought up to a final volume of 12.5 mL with
anhydrous DMF to give a stock solution of pyrophosphate with concentration of
0.4 mmol=mL. 50-Phosphonomethyl-50-deoxythymidine (213 AU260, 0.02 mmol)
was passed through a column of Dowex 50W-X2 (pyridinium form) ion-exchange
resin, then concentrated and treated with 1 equiv. of tri-n-butylamine (5.3 mL,
0.02 mmol). The nucleotide was dried by repeated co-evaporation in vacuo with
anhydrous pyridine (2 ꢁ 5 mL), and then with anhydrous DMF (3 ꢁ 10 mL). A solu-
tion of the nucleotide (0.02 mmol) in 0.5 mL of anhydrous DMF was treated with
1,10-carbonyldiimidazole (0.11 mmol). After two hours at room temperature the
mixture was treated with the pyrophosphate stock solution (275 mL, 0.11 mmol),
and stirred at room temperature for 2 days under argon. It was then filtered, and
the precipitate was washed with 1 mL of MeOH and then 1 mL of DMF. The filtrate
and the combined washings were concentrated and the products separated by paper
chromatography using solvent system B. After drying, the paper was washed with
ethanol to remove isobutyric acid, and the triphosphate analogue band was eluted
with water (yield, 41%). The product was converted to its sodium salt by passage

dTTP Analogue
409
through a column of Dowex 50W-X8 (Naþ form), and was shown to be pure by
chromatography on the PEI silica column.
Primer Extension Reactions on RNA Templates
All extension reactions were 20 mL in volume and contained 50 mM Tris-HCl
(pH 8.3), 75 mM KCl, 3.0 mM MgCl2, 1.0 mM DTT, 200 U of reverse transcriptase,
0.125 pmol of oligonucleotide primer (either unlabeled or labeled at the 50 terminal
phosphate with 32P), 0.25 pmol rabbit b-globin mRNA, 20 U of RNase inhibitor,
0.5 mM each of dATP, dGTP, and dCTP, and either 0.5 mM dTTP or 1.0 mM of
the thymidine triphosphate analogue. Prior to the addition of the enzyme, DTT,
and RNase inhibitor, each reaction mixture was heated to 70ꢂC for 5–10 min. The
complete reaction mixture was incubated at 37ꢂC for 2 h, and then heated for
10 min at 70ꢂC. A portion of each reaction mixture was mixed with an equal volume
of 90% formamide containing 0.1% xylene cyanol FF and 0.1% bromophenol blue.
Samples were heated to 70ꢂC for 10 min and subjected to electrophoresis through
15% denaturing gels for 3 h at 100 volts. Gels were dried and the labeled bands of
products were visualized by autoradiography in the usual way. In order to obtain
an approximate estimate of product chain lengths, portions of the reaction mixtures
were treated as above and subjected to electrophoresis through 11% denaturing gels
at 1700 volts alongside a set of 32P-labeled standard markers of chain length 100x
where x ¼ 1–6.
Sequence Analysis of DNA Products
The 50-labeled DNA products from the reverse transcriptase reactions described
above were purified on NAP-10 columns using procedures supplied by the manufac-
turer. The resulting samples were concentrated to dryness and then further purified
on Microcon spin columns again using the manufacturer0s protocols, and the final
solutions were evaporated to dryness in vacuo. Samples were dissolved in 10 mL of
water and subjected to depurination=b-elimination reactions as described.[8] The
cleavage products were then separated by electrophoresis on an 11% denaturing
gel at 1700 volts for 5 h.
Second Strand Synthesis Using Reverse Transcriptase
DNA copies of globin mRNA using either dTTP or its analogue were prepared
as described above except that unlabeled primer was used. Each reaction mixture
(20 mL) was then mixed with 10 mL of 0.3 M NaOH and kept at 37ꢂC for 30 min in
order to degrade the mRNA template. The alkaline mixture was then neutralized
by addition of 10 mL of 0.3 M HCl. The DNA product in each reaction mixture
was purified as described above for the sequence analysis experiments. Each chain
extension on the DNA template was performed in a volume of 20 mL containing
50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3.0 mM MgCl2, 0.125 pmol of an octa-
decanucleotide primer labeled at its 50 terminal phosphate with 32P, along with
0.5 mM each of dATP, dGTP, and dCTP, and either 0.5 mM dTTP or 1.0 mM of

410
Bell, Gough, and Gilham
the thymidine triphosphate analogue. Each reaction mixture was first heated to 70ꢂC
for 5–10 min, and then incubated at 37ꢂC for 2 h with 1.0 mM DTT and 200 U of
reverse transcriptase. Each mixture was then heated at 70ꢂC for 10 min and the pro-
ducts were either directly analyzed by gel electrophoresis as indicated above or sub-
jected to endonuclease digestion prior to analysis. Restriction endonuclease digests
were carried out by mixing 10 mL from each second strand synthesis reaction with
10 mL of a buffered solution of either EcoRI or HaeIII as described in the literature.[8]
Each digest was allowed to proceed at 37ꢂC for 30 min. and then subjected to
electrophoretic analysis on a 15% denaturing gel. Chain extensions on a chemi-
cally-synthesized template were performed with a 90-nucleotide DNA strand and
appropriate primer (both kindly provided by Dr. H. L. Weith). The conditions
and analyses were identical with those used above for templates that were produced
enzymatically.
Polymerase Chain Reactions
PCR reactions (50 mL) contained 10 mM Tris-HCL (pH 9.0), 50 mM KCl, 5 U
Taq DNA polymerase, MgCl2 (optimum concentration: 4.0 mM), 0.5 pmol each of
‘‘top’’ and ‘‘bottom’’ primers (one of which was 50 end-labeled with 32P), 0.025 mg
of template (either pUC18 or pUC18 with an insert of A-T tracts), and 20 mM each
of dATP, dGTP, dCTP, and either 20 mM dTTP or 240 mM of its analogue. The PCR
cycle, consisting of denaturation at 92ꢂC for 2 min, annealing at 45ꢂC for 5 min, and
polymerization at 70ꢂC for 10 min, was repeated 30 times. Products were analyzed by
electrophoresis on 15% gels using the conditions described above.
RESULTS
The introduction of the phosphonomethyl group into the 50 position of the deoxy-
ribonucleoside thymidine was accomplished using modifications of the procedures
described by Jones and Moffatt[9] for preparation of the corresponding ribonucleo-
tide analogues of UMP and AMP from the starting materials 20,30-O-isopropylide-
neuridine and 20,30-O-isopropylideneadenosine, respectively. For the work with
thymidine we employed acetyl to protect the 30-hydroxyl: 30-acetylthymidine was
treated with dimethyl sulfoxide and dicyclohexylcarbodiimide to yield the 50-alde-
hyde. This material was then treated with the Wittig reagent diphenyl triphenylpho-
sphoranylidenemethylphosphonate, and the resulting product was subjected to
catalytic hydrogenation to reduce the double bond. The acetyl and one of the phenyl
groups were removed by mild alkaline treatment to yield the monophenyl ester of 50-
phosphonomethyl-50-deoxythymidine, and removal of the second phenyl group was
achieved by exposure to snake venom phosphodiesterase. Finally, the pyrophos-
phate group was added using a modification of the procedure used by Hoard and
Ott[10] for conversion of deoxyribonucleoside 50-phosphates to their corresponding
50-triphosphates.
Reverse transcriptase activity was tested using rabbit b-globin mRNA as tem-
plate along with the primer pAATTTCCTTTATT labeled at its 50 end with 32P.
In the expected primer=template complex, the 50 terminal nucleotide of the primer

dTTP Analogue
411
Figure 1. Gel patterns produced by reverse transcriptase primer extensions on rabbit
b-globin mRNA. Lane 1 contained the labeled primer used. Lanes 2–6 were the products from
reactions containing 0.05 mM each of dATP, dCTP, and dGTP. The reactions in Lanes 2 and
5 contained 0.05 mM dTTP, while those in 4 and 6 contained the analogue thymidine tri-
phosphate at 1.0 mM concentration. The reactions in Lanes 2–4 were run overnight while
those in Lanes 5 and 6 were run for 2 h.
should be located opposite nucleotide #577 of the mRNA template (GenBank
Accession # V00879). Chain extension experiments with either thymidine triphos-
phate or its analogue yielded full-length transcripts of similar chain length
(Fig. 1). One of the control reactions for these chain extensions contained the three
triphosphates dATP, dCTP, and dGTP only, and while this control indicated some
activity, it is clear that no complete transcript was formed. We attribute this minor
activity either to trace amounts of dTTP contaminating one or more of the other
three triphosphates or to a low level of non-Watson Crick incorporation. Assign-
ments of the chain lengths of the two transcripts were made by electrophoresis on
a longer polyacrylamide gel (Fig. 2). The positions of the two transcripts are consis-
tent with their expected chain lengths of 577 each. Next, a partial sequence analysis
was carried out to check that the analogue thymidine residues were undergoing inser-
tion only in positions opposite the adenosine moieties in the RNA template. The
analysis of partial depurination reactions on the two transcripts produced identical
cleavage patterns upon electrophoresis, and these patterns are readily accounted for
on the basis of the expected purine positions in the transcripts (Fig. 3). For example,
the series of 13 contiguous purine cleavages that give rise to the chain length set of

412
Bell, Gough, and Gilham
Figure 2. Gel electrophoretic analysis of DNA’s made using reverse transcriptase with rabbit
b-globin mRNA as template. The reactions in Lanes 1–3 were run for 2 h and each contained
0.5 mM dATP, dCTP, and dGTP. The reaction in Lane 1 also contained 0.05 mM dTTP,
while that in Lane 3 contained 1.0 mM of the thymidine triphosphate analogue. Chain length
markers were located in Lane L.
65–77 correspond to the location of the 13 contiguous pyrimidines (at positions 500–
512) in the template.
For second strand synthesis the first series of experiments employed the set of 4
regular nucleoside triphosphates with either normal or analogue DNA as template.
The templates were prepared from globin RNA as described above, except that the
primer used was unlabeled at its 50 end (Fig. 4). The 50 labeled primer selected for the
copying reactions was assigned the sequence complementary to the area occupied by
nucleotides #168 to #185. The double-stranded products from these reactions were
expected to have recognition sites for the restriction endonucleases, HaeIII and
EcoRI, at positions #108 and #156, respectively. The results of the chain extension

dTTP Analogue
413
Figure 3. Depurination patterns produced from normal and analogue transcripts made with
rabbit b-globin mRNA as template. The left-hand pattern shows bands with chain lengths of
32 to 111 while the bands in the right-hand pattern have chain lengths of 13 to 65. Lane 1 has
products deriving from the use of dTTP in the transcription reaction and Lane 2 are those
deriving from the use of the analogue dTTP.
reactions are shown in Fig. 5. Chain extensions using either the normal DNA tem-
plate or the template prepared from the nucleotide analogue gave full-length second
strands of 185 nucleotides. Treatment of the double-stranded products with either
HaeIII or EcoRI before electrophoresis showed the expected shortened chains of

414
Bell, Gough, and Gilham
Figure 4. Sequence of DNA used for study of second strand synthesis. The primer used was
complementary to 18-nucleotide run (underlined) located at positions 168–185. The locations
of the endonuclease HaeIII site (GGCC) and EcoRI site (GAATTC) are also underlined.
Figure 5. Gel patterns derived from second strand syntheses using the four regular nucleoside
triphosphates and the labeled primer and DNA template shown in Fig. 4. The left-hand
panel contains products from the normal DNA template, while the right-hand panel shows
products using the analogue DNA as template. For each panel, Lane 1 shows the full exten-
sion reaction out to a chain length of 185; Lane 2 is the pattern obtained by treatment of the
reaction product with endonuclease EcoRI before electrophoresis; and Lane 3 is the pattern
obtained by treatment of the reaction mixture with endonuclease HaeIII.

dTTP Analogue
415
Figure 6. Chemically synthesized oligonucleotide and primer for chain extension reactions
containing the analogue thymidine triphosphate.
chain lengths 76 and 25, respectively. The observed cleavages of the duplex contain-
ing the analogue strand are worthy of note; the location of multiple phosphonate
internucleotide linkages near or within the recognition sites seemed to have little
effect on the restriction enzymes’ activities.
A second series of experiments tested the capacity of the analogue triphosphate
to serve as a substrate for second strand synthesis. However, all attempts to use this
triphosphate with the analogue DNA strand as template failed. In order to test sec-
ond strand synthesis using a natural DNA strand as template, it was necessary to
employ a template uncontaminated with normal nucleoside triphosphates. A chemi-
cally-synthesized 90-nucleotide polymer (Fig. 6) and an appropriate 32P-labeled
primer were used. Fig. 7 shows the gel patterns of the products of these extension
Figure 7. Chain extension reactions using the synthetic DNA=primer shown in Fig. 6. Lane 1
shows the gel pattern obtained for the extension reaction using all four regular nucleoside tri-
phosphates. Lane 2 is the pattern obtained for the same reaction mixture except for the omis-
sion of the dTTP. Lane 3 is the pattern for the same reaction mixture as in Lane 1 except for
the substitution of the dTTP with the analogue triphosphate.

416
Bell, Gough, and Gilham
reactions: the product containing the analogue thymidine residues matches that
obtained with the 4 regular triphosphates. Again, in this study, the control reaction
containing only the triphosphates of A, G, and C indicated traces of chain extension,
and these also can be attributed to some contamination of the commercially-
obtained monomers with dTTP.
Finally, a series of experiments were conducted to test the ability of Taq DNA
polymerase to accept the analogue in place of dTTP. PCR reactions were carried out
under a wide variety of conditions, using pUC18 plasmids as templates along with
the appropriate labeled primers. While control reactions with the 4 regular triphos-
phates yielded the expected PCR product, none was detected in reactions containing
the analogue in place of the dTTP.
DISCUSSION
In view of these results, it seems possible that exposure of a retrovirus in vivo to
the dTTP analogue could interfere with its replication cycle. Inhibition of the cycle
might occur at any of the various steps of reverse transcription, integration, and
transcription. One biochemical step that merits further detailed study is that of
second strand synthesis by reverse transcriptase. The experiments described above
indicate that the analogue-containing DNA strand can serve as template in second
strand synthesis when dTTP is present in the reaction, but not when the analogue
is the only source of thymidine. Specifically, it would be of interest to know the
effects on first and second strand syntheses when both natural and analogue triphos-
phates are present. However, a definitive study of the relative incorporation of the
two substrates will require the use of differential labels such as, for example, a-32P
in dTTP and 14C in its analogue. Another aspect that should be investigated further
is the interaction of the phosphonate analogue triphosphates with DNA-dependent
DNA polymerases. The lack of product formation in the PCR reactions with Taq
DNA polymerase may be due to complete rejection of the analogue as substrate.
Alternatively, the polymerase may be behaving in a manner similar to that of the
reverse transcriptase by incorporating the analogue during first strand formation
to generate a template that is unacceptable for second strand synthesis.
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phodiester 50-oxygens. Biochemistry 1993, 32, 9125–9128.
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dTTP Analogue
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Received October 29, 2002
Accepted February 4, 2003