STABILITIES OF NUCLEOTIDE LOOPS BRIDGING THE PYRIMIDINE STRANDS IN DNA PYRIMIDINE-CENTER-DOT-PURINE-CENTER-DOT-PYRIMIDINE TRIPLEXES - SPECIAL STABILITY OF THE CTTTG LOOP

Recent studies of DNA hairpin loops have shown considerable dependence of the stability on the sequence of the loop [Senior, M., Jones, R. A., & Breslauer, K. J. (1988a) Proc. Natl. Acad. Sci. U.S.A. 85, 6242-6246; Xodo, L. E., Manzini, G., Quadrifoglio, F., van der Marel, G., & van Boom, J. H. (1989) Biochimie 71, 793-803; Hirao, I., Nishimura, Y., Tagawa, Y., Watanabe, K., & Miura, K. (1992) Nucleic Acids Res. 20, 3891-3896]. Analogous studies have not, until now, been carried out with loops in triple helices. We report the results from experiments in which we examine the relative stabilities of pentanucleotide loops that bridge between the pyrimidine strands in DNA pyr.pur.pyr triple helices. There are two types of loops that are defined by the relative orientation of the purine strand: a 5'-loop and a 3'-loop. The sequences examined in this study are the bimolecular triplexes formed between 5'-dTTCTTTTCL(1)TTTL(5)-CTTTTCTT (loop nucleotides are underlined, and L(1) and L(5) represent varied nucleotides) and the two purine strands, 5'-dAAGAAAAG-3' and 5'-dGAAAAGAA-3'. The first and last nucleotides in the loop are varied, since stacking interactions may be strongest at these positions [Senior et al., 1988a; Senior, M., Jones, R. A., and Breslauer, K. J. (1988b) Biochemistry 27, 3879-3885], and we examine 14 sequence combinations for each loop type. Thermal denaturation studies carried out at pH 7.0 indicate considerable variation in the stabilities of these loops. In the 5'-loop series, the strongest complex is formed with the loop having the sequence 5'-CTTTG, with a T-m of 35.1 degrees C and a free energy (37 degrees C) of -8.2 kcal mol(-1). The least stable complex has the loop sequence 5'-CTTTC, with a T-m of 27.1 degrees C and a free energy of -4.8 kcal mol(-1) Similarly, in the 3'-loop series, the strongest complex is formed with the loops having the sequence 5'-CTTTG, with a T-m of 32.2 degrees C and a free energy of -6.7 kcal mol(-1). The least stable complex has the loop sequence 5'-CTTTC, with a T-m of 25.2 degrees C and a free energy of -5.0 kcal mol(-1). Several conclusions are drawn from the 28 different cases studied: (1) 5'-loops are somewhat more stable than 3'-loops, with an average difference of 0.4 kcal in free energy; (2) purines in both types of loop and at both positions are stabilizing, by an average of 0.5-0.9 kcal each, relative to pyrimidines (presumably, this is due to more efficient base stacking); (3) placement of a purine on the 5'-end of the helix is more favorable than on the 3'-end by an average of 0.6 kcal, thus favoring YTTTR loops over RTTTY loops (this is consistent with the stacking preferences seen for duplex DNA); (4) there is a special stability present in the cases where the first and last bases are complementary in the Watson-Crick sense, suggesting possible pairing interactions across the loop. This interaction adds as much as -0.8 kcal of stability to the 3'-end and -2.1 kcal to the 5'-end, relative to a C-A mismatched case, and thus leads to relatively high stability for complexes with TTTTA and especially those with CTTTG loops. A model for this pairing interaction is proposed.