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Influence of N-Acylation on the Stability of Double-Stranded Polydeoxynucleotides F. Newton Hayes,* Elizabeth Hansbury, Victoria E. Mitchell, and Terence J. Scallen Contribution ,from the Biomedical Research Group, Los Alamos ScientiJic Laboratory, University of California, Los Alamos, New Mexico 87544, and the Department of Biochemistry, School of Medicine, The University of New Mexico, Albuquerque, New Mexico 87106. Received August 12, 1970
Abstract: Thermal hyperchromism of polydeoxynucleotide mixtures has been studied relative to degree and type of purine N-acylation. Thermally labile acyl groups confuFed interpretation of melting curves until a technique of fast heating and rapid cooling gave needed reproducibility. In a series with ac6A.T hydrogen bonding, a linear relationship of T , to degree of acetylation was found. Relative stabilities of hydrogen-bonded association proved to be A . T > ac6A' T > ibsA' T and ac6A' T > acZG C. Purine acetylation in polydeoxynucleotides has made possible novel and fundamental thermal hyperchromism studies as well as allowing enzymatic chainlengthening reactions which would otherwise have been impossible due to aggregation and interfering hydrogen bonding.
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t has been observed' during a study of synthetic polydeoxynucleotides that monoacetylation of the 6-amino group of adenine sharply decreased the midpoint (T,) in the thermal hyperchromicity transition (melting curve) of the Watson and Crick2 A . T doublehydrogen bonding (Figure 1). At the same time it was reported' that melting studies on the effect of monoacetylation of guanine, presumed to be on the 2-amino group, failed to establish existence of the acetylated G . C triple-hydrogen bonding (Figure 2), since no sharp melting transitions were observed. Subsequently, Lefler and Bollum3 reported that they could obtain no evidence for definite complexes formed in physical mixtures of single-stranded polydeoxycytidylate (dC,) * with poly-N-acetyldeoxyguanylate d(ac2G),. Now in a detailed corroboration of our early observations, melting curves along with associated ultraviolet absorption data have been obtained that describe various degrees of double-helix destabilization resulting from acylation of amino groups in purines of synthetic polydeoxynucleotides.
Experimental Section Polydeoxynucleotide concentrations are expressed in phosphorus concentrations determined according to Petersen, et 01.~ Ultraviolet absorption measurements were carried out on a Unicam SP-800 spectrophotometer equipped6 for plotting of melting curves. Each T , value was determined as the temperature of maximum slope, and Amaxis the maximum slope expressed as the increase in A units per degree; both of these parameters were evaluated from calculated differential melting curves.6 Address correspondence to this author at Los Alamos Scientific Laboratory. (1) F. N. Hayes, E. Hansbury, V. E. Mitchell, R. L. Ratliff, and D. L. Williams, Eur. J. Biochem., 6 , 485 (1968). (2) J. D. Watson and F. H. C. Crick, N a m e (London), 171, 737 (1953). (3) C. F. Lefler and F. J. Bollum, J . Biol. Chem., 244, 594 (1969). (4) Abbreviations used are those of the JUPAC-IUB Combined Commission on Biochemical Nomenclature, Biochemistry, 9,4022 (1970). The substituent designations ac and ib refer to acetyl and isobutyryl, respectively. Overlining of a subscript means that the number is the average of a distribution. ( 5 ) D. F. Petersen, L. B. Cole, E. H. Lilly, and V. E. Mitchell, Los Alamos Scientific Laboratory Report No. LAMS-2780, 1962, p 300. (6) F. N. Hayes, E. H. Lilly, R. L. Ratliff. D. A. Smith, and D. L. Williams, Biopolymers, 9, 1105 (1970).
The solvents used were 0.1 M Na+, pH 7.0 (0.08 M NaCI-0.01 M Na2HP0.r,pH 7.0), 0.5 M Na+, pH 7.8 (0.48 M NaCI-0.01 M Na2HPOa, pH 7.8), 1.0 M Na+, pH 7.0 (0.98 M NaCI-0.01 M Na2HP04,pH 7.0), and 1.0 M Na+, pH 8.1 (0.98 M NaC1-0.01 M
Na2HPOa,pH 8.1). Oligodeoxynucleotides dT8, dTl;, and dCr were prepared by chemical polymerization according to Khorana and V i ~ s o l y i . ~ Using dTs and dCa as initiators with appropriate deoxynucleoside 5'-triphosphates and calf thymus terminal deoxynucleotidyltransferase? the polydeoxynucleotides dT;, dC,, d(T6-A$, d(T6-A6;,), and d(TsaC2GG) were synthesized and characterized.9 Chemical acylation*0of d ( r 6 - A ~ )and d(TE-Az)gave d(Ts-aCGA$, d(T6-ac6AG), and d(T6-ib6A~,).Ammonolysis at 26" of d(T6-ac6A:,) (66 PILI in 1.3 M aqueous NH3) reached 50% deacetylation in 13 hr. The degree of acetylation was established by comparing uv absorption, , ,A values with those obtained fiom a set of standard mixtures of nonacetylated with totally acetylated polymers. Complete acetylation was judged bq finding no change in properties on further acetylation.
Results Spectral Measurements. Mixing curves were run and analyzed according to Riley, et al.," with incremental addition of dT, t o d(TC-ac6AGj)in 1.0 M Na+, pH 7.0 at 21", of dT, to d(T6-ib6Asj)in the same solvent at 8", and of d(T6-ac2G$ to dCE in 0.5 M Na+, pH 7 . 8 at 8". Plots of e(P) against the composition of the mixture gave typical intersecting lines whose intercepts within f 15 all indicated the existence of 1 : 1 complexes. A mixture of d(T6-ac6Ajo)(39.0 p M in d(acfiA)units) and dTls (39.3 p M ) in 1.0 M Na+, pH 7 . 0 (mixture I), was spectrophotometrically analyzed at 0 and 40" as shown in Figure 3. For the 0" curve 1, A, = 263.5 nm, A z j o / A 2 f i=o 0.78, A28o/A260 = 0.67; for the 40" curve 2, A, = 268 nm, A250/A260 = 0.74, A ~ ~ o / A z ~ o = 0.78; the maximum hyperchromicity ratio (2/1) = 1.47 at 276 nm. A mixture of d(T,-A,) (40.8 p M in dA units) and dT15(39.3 p M ) in 1.0 M Naf, pH 7 . 0 (mix(7) H. G. Khorana and J. P. Vizsolyi, J . Amer. Chem. Soc., 83, 675 (1961). (8) M. Yoneda and F. J. Bollum, J . Biol. Chem., 240, 3385 (1965). (9) F. N. Hayes, V. E. Mitchell, R. L. Ratliff, A. W. Schwartz, and D. L. Williams, Biochemisrry, 5, 3625 (1966). (10) E. Hansbury, V. N. I
