Mechanism of degradation of purine nucleosides by formamide. Implications for chemical DNA sequencing procedures

Article abstract of DOI:10.1021/ja953527y

We describe the reaction of formamide with 2'-deoxyadenosine and 2'-deoxyguanosine to give imidazole ring opening by nucleophilic addition on the electrophilic C(8)-position of the purine ring. This information allows improvement of the one-lane chemical DNA sequencing procedure based on the base-selective reaction of formamide with DNA. The reactivity with formamide of several 7-deazapurine analogues (7-deaza-2'-deoxyinosine, 7-deaza-2'-deoxyguanosine, and 7-deaza-2'-deoxyadenosine) incorporated into polynucleotides is also described. The wide spectrum of different sensitivities to formamide displayed by these purine analogues provides the single-lane DNA chemical sequencing procedures with the possibility of wide-ranging signal intensity modulation and thus increased specificity.

Full text of DOI:10.1021/ja953527y

J. Am. Chem. Soc. 1996, 118, 5615-5619
5615
Mechanism of Degradation of Purine Nucleosides by
Formamide. Implications for Chemical DNA Sequencing
Procedures†
Raffaele Saladino,*,‡ Enrico Mincione,‡ Claudia Crestini,‡ Rodolfo Negri,§
Ernesto Di Mauro,§, and Giovanna Costanzo
Contribution from the Dipartimento ABAC, UniVersita` degli Studi della Tuscia,
Viterbo 01100, Italy, Centro di Studio per gli Acidi Nucleici, CNR, Roma, Italy, and
Fondazione “Istituto Pasteur - Fondazione Cenci Bolognetti”, c/o Dipartimento di
Genetica e Biologia Molecolare, UniVersita` “La Sapienza”, Roma, Italy
ReceiVed October 20, 1995X
Abstract: We describe the reaction of formamide with 2′-deoxyadenosine and 2′-deoxyguanosine to give imidazole
ring opening by nucleophilic addition on the electrophilic C(8)-position of the purine ring. This information allows
improvement of the one-lane chemical DNA sequencing procedure based on the base-selective reaction of formamide
with DNA. The reactivity with formamide of several 7-deazapurine analogues (7-deaza-2′-deoxyinosine, 7-deaza-
2′-deoxyguanosine, and 7-deaza-2′-deoxyadenosine) incorporated into polynucleotides is also described. The wide
spectrum of different sensitivities to formamide displayed by these purine analogues provides the single-lane DNA
chemical sequencing procedures with the possibility of wide-ranging signal intensity modulation and thus increased
specificity.
Introduction
analysis offers the potential of complete compression: if one
could obtain unambiguous and complete sequence information
in a single lane, then four different DNAs, each labeled with a
different fluorochrome, could be analyzed in the same electro-
phoretic lane (four fluorochromes/four DNAs). The methods
developed so far which aim to simplify the sequencing
procedures by introducing chemical alternatives to the classical
Maxam and Gilbert protocols1 have been reviewed.5-7
Methods for accurately establishing the order of the four bases
along given DNA fragments are based on two different
principles. One method uses chemical reagents that react with
specific bases to break DNA preferentially at given nucleotides,1
the other is based on the analysis of the products of DNA
polymerization selectively interrupted with chain terminating
deoxyribonucleotides.2
The DNA sequencing procedure based on the selective
degradation of nucleic acids by formamide provides complete
sequence information in a single electrophoretic lane, by
measuring intensity of the signal corresponding to each cleaved
sequence position.8-11 This procedure depends on the degrada-
tion of the purine and pyrimidine bases by formamide at high
temperature (>100 °C) followed by scission of the glycosidic
linkages through â-elimination reactions. In the presence of a
weak base such as formamide, only the 3′-â-elimination occurs;8
efficient â-elimination at 5′ (leading to unbiased sequencing of
5′-labeled DNAs) requires the use of piperidine9,10 as a second
reaction step. Irrespective of the specific protocol used for the
phosphodiester bond breakage, the selective (i.e., base-specific)
part of the reaction is carried out by formamide.8-11
The ideal DNA sequencing method should yield unambiguous
and complete information in a single electrophoretic lane and
should be simple, rapid, economical and accurate. Compression
of DNA sequencing procedures is crucial for development and
improvement of automated analytical systems. Methods for
partial sequence data compression have been reported,3,4 and a
single-lane sequencing procedure is currently available, based
upon the dideoxy Sanger methodology, analyzed in automated
sequences. This method (four fluorochromes/four bases) cannot
a priori be compressed further. Chemical DNA sequencing
* Corresponding author: Raffaele Saladino, Dipartimento ABAC Uni-
versita` degli Studi della Tuscia, Via Camillo De Lellis, Viterbo 01100, Italy.
Tel. + 39.761.357230. Fax. + 39.761.357242.
‡ Universita` degli Studi della Tuscia.
§ Centro di Studio per gli Acidi Nucleici.
In any sequencing protocol, the best analytical condition is
one in which the difference in reactivity between the four bases
is homogeneous and large. The order of sensitivity observed
in the formamide reaction is G > A > C . T, where the
reactivity ratio Gs/As ranges from 1 to 1.5 depending on the
Universita` “La Sapienza”.
† Abbreviations: G, guanosine; A, adenosine; I, inosine; C, cytidine; T,
thymidine; ATP, adenosine triphosphate; CTP, cytidine triphosphate; dATP,
2′-deoxyadenosine triphosphate; dGTP, 2′-deoxyguanosine triphosphate;
dITP, 2′-deoxyinosine triphosphate; deazaA, 7-deaza-2′-deoxyadenosine;
deazadATP, , 7-deaza-2′-deoxyadenosine triphospate; deazadGTP, 7-deaza-
2′-deoxyguanosine triphosphate; deazadITP, 7-deaza-2′-deoxyinosine tri-
phosphate; PCR, polymerase chain reaction; PE, primer extension; TLC,
thin layer chromatography; EDTA, ethylenediaminetetraacetic acid; BSTFA,
N,O-bis(trimethylsilyl)trifluoroacetamide.
(5) Ambrose, B. J. B.; Pless, R. C. Methods Enzymol. 1987, 152, 522-
538.
(6) Testoff, M. A.; Pless, R. C. Anal. Biochem. 1991, 197, 316-320.
(7) Rosenthal, A. Methods in Molecular Biology; Human Press: New
York, 1993;Griffin, H., & Griffin, A., Eds.; Vol. 23, pp 261-280.
(8) Negri, R.; Costanzo, G.; Di Mauro, E. Anal. Biochem. 1991, 197,
389-395.
X Abstract published in AdVance ACS Abstracts, June 1, 1996.
(1) Maxam, A. M.; Gilbert, W. Proc. Natl. Acad. Sci. US.A. 1977, 74,
560-563.
(2) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A.
1977, 74, 5463-5467.
(9) Ferraboli, S.; Negri, R.; Di Mauro, E.; Barlati, S. Anal. Biochem.
1993, 214, 566-570.
(3) Nelson, M.; Zang, Y.; Steffens, D.; Grabherr, R.; van Hetten, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1647-1651.
(10) Negri, R.; Ferraboli, S.; Barlati, S.; Di Mauro, E. Nucleic Acid Res.
1994, 22, 111-112.
(4) Ansorge, W.; Zimmermann, J.; Schwarger, C.; Stegemann, J.; Erfle,
H.; Voss H. Nucleic Acids Res. 1987, 18, 3419-3420.
(11) Di Mauro, E.; Costanzo, G.; Negri, R. Nucleic Acids Res. 1994,
22, 3811-3812.
S0002-7863(95)03527-X CCC: $12.00 © 1996 American Chemical Society

5616 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Saladino et al.
reaction conditions; the reactivity ratio Cs/As is 0.15, and the
reactivity of Ts is close to 0. The variations on the basic
protocols used to solve these biases have been described.8-11
In order to simplify the formamide sequencing procedure and
to abolish the possibility of errors in the determination of Gs
and As residues, we have found the use of purine derivatives
to be quite promising. These could (i) be incorporated into the
DNAs to be analyzed by either the polymerase chain reaction
(PCR)12 or primer extension (PE)7 procedures and (ii) be
degraded at different rates, in order to increase the sensitivity
intervals.
Knowledge of the degradation pathway of purine nucleosides
with formamide is necessary to select analogues to be incor-
porated and then selectively degraded. Although the degradation
pathway of purine nucleosides and nucleotides by the action of
strong bases (usually HO-) is widely described in literature,13
the reaction with a weak base such as formamide has not been
previously studied. In this paper we describe the mechanism
of degradation of 2′-deoxyadenosine (1) and 2′-deoxyguanosine
(2) with formamide. The degradation pattern of purine nucleo-
sides, as defined by this analysis, allows us to predict the
positions of the purine ring whose structural modifications may
modify the sensitivity toward formamide, therefore allowing
substantial improvement of the DNA chemical sequencing
procedure.
previously described for efficient nucleophilic agents, such as
hydrazine and hydroxylamine.16
According to the degradation pathway hypothesized (Scheme
1) the initial C(8)-adduct between formamide and 2′-deoxy-
adenosine (which could not be isolated, probably because of
its instability) may easily undergo the C(8) imidazole ring-
opening to yield the derivative 3. Compound 3 is probably the
first to form and it might be slowly transformed to 4 by loss of
the initially acquired molecule of formamide. Under sufficiently
vigorous conditions (i.e, removal of the formamide by distillation
under vacuum) the 2′-deoxyribosyl residue may be hydrolyti-
cally detached to yield compound 5.
It is noteworthy that in the reaction of 1 with formamide
products of possible alternative degradation pathways (namely,
nucleophilic additions at the C(6) and/or C(2)-positions on the
purine ring) are not recovered in the reaction mixture.
2′-Deoxyguanosine (2) reacts faster than 1 with formamide
at 110 °C, in agreement with the order of sensitivity observed
for the purine bases in DNA.8-11 The reaction was complete in
only 3 h resulting in a complex mixture that was not character-
ized further. When the reaction was performed at 90 °C a minor
conversion (15%) of the substrate was observed. In the latter
case, 2,6-diamino-4-oxo-5-formamido-6-[N-(2′-deoxy-â-D-ri-
bofuranosyl)]pyrimidine 6 and 2,6-diamino-4-oxo-5-formami-
dopyrimidine 7 were obtained (Scheme 2) after purification by
TLC in reverse phase (butanol saturated by water) or by flash-
chromatography (after distillation of the excess formamide).
Formamidopyrimidine derivatives (FAPyr) 4, 5, 6, and 7 are
usually referred to as products of imidazole C(8)-ring cleavage
of purine nucleosides and nucleotides by ionizing radiation17-20
or by basic treatment after heterocyclic nitrogen alkylation.21
On the basis of the structural analogies among the isolated
degradation products it is reasonable to suggest that the
degradation pathway of 2′-deoxyguanosine is similar to that
described for 2′-deoxyadenosine, even if, in the first case, other
degradation pathways cannot be completely excluded because
of the lack of characterization of all possible intermediates.
Results
In order to obtain information on the degradation pathway
of purine nucleosides by formamide, we have analyzed the
reactions of 2′-deoxyadenosine (1) and 2′-deoxyguanosine (2)
using the experimental conditions described for the formamide
DNA sequencing method.11 2′-Deoxyadenosine (1) (538 mg,
2 mmol) was added to formamide (10 mL) in the presence of
low amount of water (0.1 mL), and the mixture was refluxed at
110 °C until the disappearance of the substrate. Small amounts
of the formamidopyrimidine nucleoside 3 and 6-amino-5-
formamido-4-[N-(2′-deoxy-â-D-ribofuranosyl)]pyrimidine 4
(Scheme 1) were obtained in isolated form by analytical TLC
purification in reverse phase (butanol saturated with water) and
characterized by capillary gas chromatography-mass spectrom-
etry (CG-MS) after silylation with N,O-bis(trimethylsilyl)-
trifluoro acetamide (BSTFA),14 1H-NMR, and elemental mi-
croanalysis. Compound 3 was found to be unstable even if
recovered under nitrogen atmosphere, and it was easily con-
verted to 4 by mild warming or by treatment with acidic SiO2.
The remaining reaction mixture was distilled under high vacuum
to eliminate the excess formamide, and the crude product was
purified by flash-chromatography (chloroform:methanol ) 9.5:
0.5 as eluent) to give 4,6-diamino-5-formamidopyrimidine 5 as
the only recovered product. These data suggest that formamide
reacts with 1 cleaving the adenine base through nucleophilic
attack on the C(8) position of the purine ring. The attack on
this position is in agreement with its electrophilic character15
and with the chemical behavior toward DNA purine components
The indication that formamide degrades purine nucleosides
by C(8)-imidazole ring-opening may provide the chemical clues
to predict the variation of reactivity for various purine analogues.
In fact, structural modifications that reduce the electrophilic
character of the C(8)-position of the purine ring might reduce
its reactivity toward a weak nucleophile such as formamide.
Available 7-deaza-2′-deoxypurine nucleosides, which lack the
electron-withdrawing nitrogen at the adjacent C(7) position,
satisfy this condition and might support nucleophilic attack less
efficiently. In order to test this hypothesis and to apply this
rationale to the chemical DNA sequencing procedure, we have
analyzed the sensitivity to formamide of 7-deazaanalogues of
purine nucleosides (both adenosine, guanosine and inosine)
when inserted into polynucleotides.
Figure 1 shows in panel a the molecular construct used to
synthesize double stranded DNA molecules carrying 7-deaza-
purine residues. The indicated 34mer template segment (lower
strand) was annealed to the 16mer oligo (upper strand) and used
as a template-primer substrate for a Sequenase-driven polym-
erization. The resulting upper strand contains (outside the oligo)
(12) Erlich, H. A. PCR Technology: Principles and application for DNA
amplification; Stockton Press: 1989.
(13) (a) Mian, A. M.; Walker, R. T. J. Chem. Soc. 1968, (C), 2577. (b)
Price, C. C.; Gache, G. M.; Koneru, P.; Shibakawa, R.; Sawa, J.R.;
Yamaguchi, M. Biochim. Biophys. Acta 1968, 166, 327. (c) Lawley, P. D.;
Brookes, P. J. Mol. Biol. 1967, 25, 143. (d) Townsed, L. B.; Robins, R. K.
J. Am. Chem. Soc. 1963, 85, 247. In Synthetic procedures in nucleic acid
chemistry; Zorbach, W. W., Tpson, R. S., Eds.; Wiley: New York-London,
1968; p 315. (e) Magrath, D. I.; Brown, G. B. J. Am. Chem. Soc. 1957, 79,
3245. (f) Gordon, M. P.; Weliky, V. S.; Brown, G. B. J. Am. Chem. Soc.
1957, 79, 3245. (g) Lawley, P. D. Proc. Chem. Soc. 1957, 290.
(14) Ali, S. L. Chromatographia 1975, 8, 33-39.
(16) Verwoerd, D. W.; Zillig, W.; Kohlage, H. Z. Physiol. Chem. 1963,
332, 184-193.
(17) Fornace, A. J.; Little, J. B. Biochim. Biophys. Acta 1977, 477, 343-
355.
(18) Ward, J. F. Isr. J. Chem. 1972, 10, 1123-1138.
(19) Phillips, G. O.; Criddle, W. J. J. Chem. Soc. 1962, 2740-2744.
(20) Chetsanga, C.; Grigorian, C. Proc. Natl. Acad. Sci. U.S.A. 1985,
82, 633-637.
(15) Pullman, B.; Jortner J. Molecular basis of specificity in nucleic
acids-drug interactions; Kluwer Academic: Dortrecht, 1961.
(21) Tabor, S.; Richardson, C. C. J. Biol. Chem. 1989, 264, 6447-6450.

Degradation of Purines and DNA Sequencing
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5617
Figure 2 shows in panel a the molecular construct used to
synthesize double stranded DNA molecules carrying G-substi-
tuted residues. The template-primer system used is similar to
the one used for the analysis of the A-analogues, except for the
modifications at positions number 22, 28, and 34 (see Experi-
mental Section). The resulting upper strand contains (outside
the oligo) sites which incorporate G residues only at the
positions 22 and 28 and was synthesized using alternatively
dGTP, dITP, deazadGTP, or deazadITP.
The reaction of four differently substituted polynucleotides
with formamide in standard conditions (110 °C, 10 min) was
analyzed on a sequencing gel (Figure 2 panel b). Panel c
compares the reactivity of the substituted residues.
The results show that the different purine analogues tested
display a remarkable difference of sensitivity to formamide
attack. The following order of sensitivity is noted: I > G >
deazaG > deazaI (I > G has already been reported).11 It is
noteworthy that also in this case 7-deaza modification strongly
decreases susceptibility to formamide attack, to the point that
deazaI is essentially inert.
Note that in the part of the molecule used as primer in this
experiment (upper part of the gel lanes), G residues are present.
Therefore, in the case of molecules containing substitutions, the
reactivity of guanosine can be directly compared in the same
molecule with that of the substituted residue. In conditions of
over-reaction (at longer times), the specificity of the sequencing
reaction tends to be lost (i.e., T residues become erratically
cleaved).
The small difference observed in the cleavage sensitivity of
As and Gs residues upon formamide attack is a potential source
of errors in the formamide-based DNA sequencing methods.8
This problem may be addressed by increasing the sensitivity at
G positions by incorporating inosine in the corresponding
positions in the PCR-amplified DNA to be sequenced.11 An
alternative, simpler solution is offered by the present observation
that deazaA residues are less sensitive toward formamide
degradation than A residues. Given that the incorporation rate
of deazaA’s is very similar to that of A’s and that deazaA’s do
not cause chain termination (not shown), the G/A difference
can be increased by incorporating a fraction (say 30%) of
deazaA’s instead of A’s in the appropriate positions. This is
achieved simply by amplifying the DNA to be analyzed with a
mixture of triphosphates in which deazaAs and As are present
in the appropriate concentrations.
Figure 1. Degradation of 3′-labeled DNA by formamide. Comparison
of the reactivity of adenosine and deazaadenosine. Panel a (top): the
DNA construct used to insert the base-analogue in two defined locations
(as indicated in the lower molecule, see text). The scheme of the
procedure used for the analysis described in the following panels is
indicated. * ) labeled position. Panel b (bottom): gel analysis (18%
acrylamide, 7 M urea) of the product of the attack by formamide on
the DNA carrying substitutions. Each group of samples shows the
products of formamide degradation of molecules in which adenosine
(A) or 7-deazaadenosine (deazaA) were incorporated at positions 22
and 28, as indicated in scheme in panel a. Reactions were carried out
for 0, 15, 30, and 60 min, as indicated. Left panel: adenosine; right
panel: 7-deazaadenosine.
Experimental Section
General Procedures. 2′-Deoxyguanosine, 2′-deoxyadenosine (Al-
drich, Co.), 7-deaza-2′-deoxyinosine-, 7-deaza-2′-deoxyguanosine-, and
7-deaza-2′-deoxyadenosine triphosphates (Boehringer) were used with-
out further purification. All solvents used were ACS reagent grade
and were redistilled according to standard procedures. Formamide was
from IBI, piperidine (puriss. p.a.) from Fluka.
1H-NMR spectra were recorded with a Bruker AC-200 spectrometer
(1H frequency: 200.13 MHz). Data processing was performed with
an Aspect 3000 computer using UXNMR software. Probe 5-mm was
as follows: solvent DMSO-d6 (Aldrich); temperature, 273 K.
The application of capillary gas chromatography-mass spectrometry
(GC-MS) to chemical characterization of altered DNA bases is well
established.22,23 The relative nonvolatility of purine derivatives was
improved by the use of 2′-deoxy-3′,5′-di-O-trimethylsilyl-â-D-ribosyl
derivatives. Gas-chromatographic analysis and mass spectra were
performed on samples derivatized with BSTFA14 in pyridine at 25 °C
by use of a HP GC-MS 5972 spectrometer equipped with a HP-5MS
column (0.25 mm, 30 m). The spectra were recorded at 70-250 °C
sites which incorporate A residues only at the positions 22 and
28 and was synthesized using alternatively dATP or deazaATP.
The reaction of the two differently substituted types of
polynucleotides with formamide in standard conditions (110 °C,
10 min) was analyzed on a sequencing gel (Figure 1, panel b).
The results show that 7-deaza modification abolishes almost
completely the susceptibility to formamide attack. The sensitiv-
ity to formamide of deazaG and deazaI was analyzed with the
same approach.
(22) Dizdaroglu, M. Anal. Biochem 1985, 144, 593-603.
(23) Dizdaroglu, M.; Bergtold, D. S. Anal. Biochem. 1986, 156, 182-
188.

5618 J. Am. Chem. Soc., Vol. 118, No. 24, 1996
Saladino et al.
Figure 2. Comparison of the reactivity of guanosine, 7-deazaguanosine, inosine, and 7-deazainosine. Panel a (top): the DNA construct used to
insert G analogues in programmed locations (positions 22 and 28, as for the construct used for A analogues). Panel b (middle): gel analysis (as in
Figure 1) of the product of formamide degradation on DNAs carrying substitutions. The leftmost lane shows a whole-sequence ladder obtained by
Cu-phenantroline reaction.8,25 Each group of samples shows the products of formamide degradation of molecules in which guanosine (G),
7-deazaguanosine (deazaG), inosine (I), or 7-deazaguanosine (deazaG) were incorporated at positions 22 and 28. Reactions were carried out for 0,
15, 30, and 60 min, as indicated in the first panel. For the inosine-containing DNA, only 1/3 of the sample was loaded on the gel, to avoid
overexposure effects due to the higher reactivity of the inosine residues.11 Panel c (bottom): cleavage at the residues 22 and 28, reported as %
(ordinate) of the total degradation products, as a function of the reaction time (abscissa).
Scheme 1
Scheme 2
Reverse phase TLC was performed using “Merck 15682 RP-8 F254S”
glass sheets (butanol saturated with water). Direct phase TLC was
performed with glass sheets “Merck silica gel 60 F254” (9.5:0.5
chloroform: methanol).
Degradation of 2′-deoxyadenosine (1) and 2′-Deoxyguanosine (2)
with Formamide. General Procedure. 2′-Deoxypurine nucleoside
(2 mmol) was dissolved in formamide (10 mL) and then heated at 110
°C (90 °C in the degradation of 2) until disappearance of the substrate.
A small portion (0.5 mL) of the crude reaction mixture was analyzed
by capillary gas chromatography-mass spectrometry (GC-MS) after
silylation with BSTFA and directly chromatographed on phase-reverse
analytical thin layer plates using water-saturated butanol as eluent. The
remaining mixture was evaporated under high vacuum to dryness, and
the residue was dissolved in chloroform and purified by flash-
chromatography (chloroform-methanol ) 9.5:0.5).
with the following program: isothermal at 70 °C for the first 1 min,
then 10 °C/min, and finally isothermal at 250 °C for 40 min. Mass
spectra were recorded on a Kratos MS80 spectrometer. Melting points
were obtained on a Reichert Kofler apparatus and are uncorrected.
Elemental microanalyses were recorded on a Carlo Erba elemental
analyzer EA 1108 and all were within 0.4% of calculated values for
carbon, hydrogen, and nitrogen.
Formamidopyrimidine Derivate 3. Mp 177-180 °C (with de-
composition). IR (KBr) 3351 (OH), 3202 (NH), 2825 (CHO), 1678
(CO), 1637 (CdC), 1280, 970 cm-1
.
1H NMR (Me2SO-d6) δ 2.05-
2.25 (m, 2, H-2′, 2′′), 4.62-4.83 (m, 3, H-4′, 5′, 5′′), 4.89-5.21 (m, 1,
H-3′), 5.40-5.87 (m, 2, H-1′, and CHOH), 6.74-6.85 (br s, 4, NH),
7.70-7.81 (m, 1, CHO), 8.25 (s, 1, H-2). Mass spectrum after silylation
(24) Denayer, R. Bull. Soc. Chim. Fr. 1962, 1358-1364.
(25) Kuwabara, M. D.; Sigman, D. S. Biochemistry 1987, 26, 7234-
7238.

Degradation of Purines and DNA Sequencing
J. Am. Chem. Soc., Vol. 118, No. 24, 1996 5619
with BSTFA m/e: 458 (M+ as dimethylsilyl derivative), 413 (M - 45
[3 Me]), 384 (M - 74 [HSi(Me)3]), 369 (M - 89 [NHCHOHNHCHO]),
311 (M - 147 [HSi(Me)3 and Si(Me)3]). Anal. Calcd for
C11H19N6O5: C, 42.04; H, 5.77; N, 26.74. Found: C, 42.09; H, 5.78;
N, 26.79.
formamide is reported in Figures 1 and 2. We describe here the
procedure used in the preparation of the four DNA molecules used for
the analysis of the G-analogues (the experiment reported in Figure 2).
A similar procedure was used for the analysis of A-analogues. The
four polynucleotides segments were synthesized as follows: 260 pmol
of the 34mer template (see Figure 2, panel a) were mixed with 260
pmol of 16mer primer in 50 mM Tris HCl (pH 7.5), the annealing
mixture (20 µL) was heated 5 min at 95 °C, then slowly cooled to 30
°C and divided in four aliquots. Each aliquot was incubated 30 min at
37 °C with 0.2U of Sequenase21 (purchased from USB) in a 10 µl final
volume of 20 mM Tris HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 250
µM TTP, and 1 µM [R-32P] dATP (3000Ci/mM) and one of 250 µM
dGTP, 250 µM deazadGTP, 500 µM dITP, and 500 µM deazadITP.
The reaction was stopped by the addition of 20 µL of 90% formamide-
10 mM EDTA, heated 2 min at 90 °C, and directly loaded on a 6%
denaturing polyacrylamide gel for purification of the labeled 34mer
(see Figure 2, panel a, upper strand). End-labeling was achieved by
terminal selective incorporation of [R -32P] ATP at the 3′ extremity in
the unique A position (indicated by a star in Figure 2, panel a). A
slightly different construct was used to assay for the sensitivity of
7-deaza-2′-deoxyadenosine relative to 2′-deoxyadenosine. This con-
struct is composed of the same primer (upper strand) and of the same
template (lower strand) as the one used for the analysis of the
G-analogues, varying only for the presence of two T residues (instead
of the Cs, as indicated in Figure 1, panel a) in position 22 and 28, thus
allowing incorporation of As or deazaAs upon elongation of the upper
strand (Figure 1). The Km of Sequenase for A or deazaA does not
vary appreciably (data not detailed). Another variation consists in the
substitution of the last position (no. 34) of the template strand (from A
to G), thus allowing specific terminal labeling of the elongated strand
using [R-32P] labeled CTP.
6-Amino-5-formamido-4-[N-(2′-deoxy-â-D-ribofuranosyl)] Pyrim-
idine 4. Mp 158-160 °C. IR (KBr) 3351 (OH), 3202 (NH), 1680
(CO), 1637 (CdC), 1280, 970 cm-1 1H NMR (Me2SO-d6) δ 2.20-
.
2.85 (m, 2, H-2′,2′′), 3.30-3.95 (m, 4, H-3′,4′,5′,5′′), 6.09-6.15 (m,
1, H-1′), 6.71-7.15 (br s, 4, NH), 8.18-8.25 (br s, 1, CHO), 8.20 (s,
1, H-2). Mass spectrum after silylation with BSTFA m/e: 413 (M+ as
dimethylsilyl derivative), 358 (M - 55 [HCN and CO]), 339 (M - 74
[HSi(Me)3]), 311 (M - 102 [HSi(Me)3 and CO]), 265 (M - 148 [2
HSi(Me)3]). Anal. Calcd for C10H15N5O4: C, 44.61; H, 5.61; N, 26.0.
Found: C, 44.69; H, 5.62; N, 26.30.
4,6-Diamino-5-formamidopyrimidine 5. Compound 5 was identi-
cal to that reported by Denayer.24 Mp 224-226 °C. IR (KBr) 3215
(NH), 1677 (CO), 1637 (CdC), 1280, 970 cm-1 1H NMR (Me2SO-
.
d6) δ 6.80-7.12 (br s, 5, NH and NH2), 8.05-8.13 (br s, 1, CHO),
8.11 (s, 1, H-2). Mass spectrum m/e: 153 (M+), 124 (M - 29 [CHO]),
109 (M - 44 [NHCHO]). Anal. Calcd for C5H7N5O: C, 39.21; H,
4.60; N, 45.73. Found: C, 39.26; H, 4.57; N, 45.79.
2-Amino-4-oxo-5-formamido-6-[N-(2′-deoxy-â-D-ribofuranosyl)]-
pyrimidine 6. Mp 180-185 °C (with decomposition). IR (KBr) 3360
(OH), 3202 (NH), 1690 (CO), 1637 (CdC), 1280, 970 cm-1 1H NMR
.
(Me2SO-d6) δ 2.28-2.85 (m, 2, H-2′, 2′′), 4.05-5.20 (m, 4, H-3′,4′,5′,5′′),
5.30-5.48 (m, 1, H-1′), 6.50-6.70 (br s, 2, NH2), 6.85 (br s, 1, NH),
7.89-7.95 (br s, 1, CHO). Mass spectrum m/e: 285 (M+), 256 (M -
29 [CHO]), 241 (M - 44 [NHCHO]). Anal. Calcd for C10H15N5O5:
C, 42.10; H, 5.30; N, 24.55. Found: C, 42.03; H, 5.29; N, 24.67.
2,6-Diamino-4-oxo-5-formamidopyrimidine 7. Compound 7 was
identical to that reported by Chetsanga and Grigorian.20 IR (KBr) 3230
(NH), 1780 (CO), 1679 (CO), 1640 (CdC), 1290, 970 cm-1. Mass
spectrum m/e: 169 (M+), 140 (M - 29 [CHO]), 125 (M - 44
[NHCHO]), 82 (M - 67 [NHCHO and HCNO]). Anal. Calcd for
C5H7N5O2: C, 35.50; H, 4.17; N, 41.40. Found: C, 35.55; H, 4.16;
N, 41.38.
Acknowledgment. This work was supported by P. F.
Ingegneria Genetica (C.N.R., Italy), by G. P.d’Ateneo (Uni-
versita` di Roma, Italy), and by MURST. We gratefully
acknowledge the revision by M. Tudor and J. Amatruda.
The Formamide Sequencing Protocol. The DNA sequencing
procedure used on 3′-radioactively labeled DNA fragments is the
“formamide protocol” performed as previously described,8 consisting
essentially of heating DNA in formamide at 110 °C without further
handling. Labeling, chemical treatment, and gel analysis, were
performed as described.8 The nucleotide sequence of the DNA
fragments and the strategy used for the insertion of the 7-deaza
derivatives into polynucleotides and for testing their sensitivity to
Supporting Information Available: A detailed description
(table of fragmentation, capillary gas-chromatographic profile,
and schemes of fragmentations) of the reaction between 2′-
deoxyadenosine and formamide (6 pages). Ordering information
is given on any current masthead page.
JA953527Y