Thermodynamic studies of base pairing involving 2,6-diaminopurine

Volume 16 Number 11 1988 Nucleic Acids Research Tbermodynamk studies of base pairing involving 2,6-diaminopurine Chaejoon Cheong and Ignacio Tinoco.Jr and 'Andre' Chollet Department of Chemistry and Laboratory of Chemical Biodynamics, University of California, Berkeley, CA 94720, USA and Kjlaxo Institute for Molecular Biology S.A., Route des Acacias 46, 1211 Geneva 24, Switzerland Received February 5, 1988; Revised and Accepted May 8, 1988 Implications of these results for design of DNA oligonucleotide probes are discussed. INTRODUCTION 2,6-diaminopurine (2-aminoadenine, D) is an adenine analog which can be found in S-2L cyanophage DNA where 2,6-diaminopurine is completely substituted for adenine (1,2). Thus, DNA containing D can be transcribed into RNA. Furthermore, studies on DNA transcription showed that 2,6-diaminopurineribosyl 5'-triphosphate (DTP) was incorporated into RNA and behaved as a true analog of ATP (3). 2-aminoadenosine (rD) and 2-amjno-2'-deoxyadenosine (dD) are thought to have three hydrogen bonds with rU and dT respectively (3, 4-8). Thermodynamic studies on rD (4,5,913) and dD (6,14-17) show that substitutions of rA by rD and dA by dD thermally stabilize the duplexes significantly. For example, the melting temperature (Tm) of poly(rD):poly(rU) is about 33° C higher than that of poly(rA):poly(rU) in 1.0 M Na+ (5). For dD, the increment in Tm is less than that for rD but still high. The Tn of poly(dD):poly(dT) is 8 ~ 12°C higher than that of poly(dA):poly(dT) in 0.1 M Na + (6,14). Howard et al. obtained good agreement with the observed elevation of Tm for S-2L DNA (6) (68.7% GC content) (1,2) using this increment in r m (observed ATra = 3.6°C, estimated ATm = s.TC). A proton NMR study of d(T-D-T-D) using the nuclear Overhauser effect shows that the solution conformation of the duplex appears to be a right-handed B-form quite similar to that of duplex d(T-A-T-A) (17). Also, an NMR study of poly(dD-dT) shows a B to A transition in 4 M NaCl (7), and a crystallographic study of d(CDCGTG) shows that the molecule adopts © IRL Press Limited, Oxford, England. 5115 ABSTRACT The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the Dcontaining standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dCjDGvdCjTGj, as stable as the A:T in dCT,DTjG:dCA3TA3G, and less stable than the A:T in dCA,DA,G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCAjXAjG:dCT3YT3G is: (A:T)>(T:D)~(D:T)>(T:A)>(C:D)~(D:A)~(D:G)>(D:Q=(G:D)~(D:D)>(A:D). Nucleic Acids Research a Z-form in 33 mM sodium cacodylatc buffer (pH 6.0), 25 mM MgClj, and 10% 2-methyl2,4-pentanediol (8). Hybridization using synthetic DNA oligonucleoades is a widely used method to identify and isolate a gene whose sequence has been deduced from the amino acid sequence. In determining the sequence of the synthetic DNA probe complementary to a region of the gene, an ambiguity problem in the genomic DNA sequence arises due to the redundancy of the genetic code. This is not a trivial problem considering that a single mismatch can lower the melting temperature by as much as io°C (18-20). Table I. Van't Hoff Thermodynamic Values for Double Helix Formation of 2,6-diaminopurine Containing dCAjXAjG+dCTjYTjG in IMNaCI, pH7. A(7°,25°C X:Y A:TT:D D:T T-A-•_/ C:D D:A D:G D:C G:D D:D A:D (kcaL/mol)" -9.6 -8.8 -8.7 -as -7.8 -6.0 -5.9 -5.9 -5.6 -5.6 -5.6 -5.2 Aff° (kcal/mol)4 -68 -59 -64 -59 -59 -56 -55 -51 -61 -51 -51 -49 A5° (cal/degmol) -196 -169 -185 -168 -172 -168 -164 -152 -185 -153 -153 -147 e Tm{°C)d CT = too^M 45° 43° 41° 41* 37° 28° 27° 27° 26° 26° 26° 23° •Estimated precision in A C ° is ±0.2 kcal/mol. More significant figures of A H ° and AS° are needed to reproduce the same values of AG° reported here. 'Estimated precision in A£f° is ±3 kcal/mol. "Estimated precision in AS° is ±8 cal/degmol. 'Estimated precision in rm is ±1°. •Data from reference 20. . Data from reference 34. 5116 Many methods have been introduced to solve this problem: simultaneous screening with all the possible complementary sequence mixtures (21,22); eliminating the preferential melting of A:T versus G:C base pairs using tetramethylammonium chloride (23); considering statistical presence of bases in degenerate codons (24); using a long and unique synthetic DNA probe (2527). Recently, a new method, using probes carrying deoxyinosine (dl) with the base analog, hypoxanthine, at positions corresponding to ambiguous nucleotides, was introduced to decrease the discriminative nature of the probes among ambiguous bases (28,29). To investigate the usefulness of dl in the design of probes, thennodynamic studies were carried out by Martin et al. (30). It was known that dl has advantages in reducing specificity of DNA probes at A/C and G/C two-fold ambiguities and at four-fold ambiguities. Nucleic Acids Research Other nucleotides which are less discriminative than dl and can make stable base pairs with ambiguous genomic nucleotides deserve study. 2,6-diaminopurine (D) is a possible candidate as a base analog to probe positions of sequence ambiguity. We have measured the stabilities of a set of deoxyoligonucleotide duplexes containing each of the four normal DNA bases paired with D. The contributions of dD base pairs to duplex stability have been calculated. Also, the utility of dD in probes at positions of base ambiguity has been considered by comparing the results of this work with previous work on oligonucleotides containing only normal bases (20) and with hypoxanthine (30). MATERIALS AND METHODS The extinction coefficient of dD was found to be 6.86 mM"1 cm" 1 at the wavelength of 260 nm by dry weight For the calculation of extinction coefficients of deoxyoligonucleotides containing dD, we considered hypochromicity due only to the nearest-neighbor interactions and used the average hypochromicity of normal base nucleotides for that of dD with its nearest neighbor nucleotides. The calculations and analysis of the data followed the methods used and described in previous papers (20,30), except that we used individual double-strand baseline slopes for each melting curves to correct for the aggregation at the higher concentrations. The strand concentrations of the molecules range from 10 /iM to 1,000 /iM for dCA3XA3G:dCT3YT3G (X,Y=A,C,G,T, or D) and from 10 ^M to lOO^M for dCjXGvdQTG, (X=A or D). Table II. Van't Hoff Thermodynamic Values for Double Helix Formation of dC 3 XG 3 :dd,YG 3 in 1M NaCI, pH 7. AC°,25OC X:Y D:T A:T« (kcal/mol)-10.2 -9.3 AH" 0 (kcal/mol) -57 -52 AS' (cal/degmol) 0 Tm(°C)d CT = iOOfiM -156 -143 52° 49° "Estimated precision in AG° is ±0.2 kcal/mol. 'Estimated precision in AH° is ±4 kcal/mol. "Estimated precision in A (...truncated)