Hl

Anti-parallel binding: Purine motif

Fig. 12. Recognition motifs for triplex forming oligonucleotide binding to DNA. Note the requirement for protonation of cytosine (at N3) in the pyrimidine motif. W-C, Watson-Crick hydrogen bonds; H, Hoogsteen hydrogen bonds; RH, reverse Hoogsteen hydrogen bonds. (Reprinted from ref. 46 with permission from Oxford University Press.)

formed by Watson-Crick base pairing binds to a polypyrimidine strand in the major groove. The second polypyrimidine strand binds to the duplex through Hoogsten base pairing between thymine and A:T base pairs and between protonated cytosine and G:C base pairs, giving rise to (T*A:T) and (C+*G:C) triplets, respectively. However, because the protonation of cytosine in the third strand is an essential requirement, triplex formation involving these triplet sequences can only occur at the acidic pH of 5.6-6.0. The hydrogen bonding scheme for triplex formation is shown in Fig. 12.

The association of three strands of DNA increases the negative charge density, and hence a higher level of positive charges is required to stabilize the triple helix. In general, triplex DNA is stabilized by cation concentrations comparable to or higher than their physiological level. For example, 150 mMNaCl or 25 mMMgCl2 can stabilize triplex structures in poly(dA)/2poly(dT). Hampel et al. (47) demonstrated that triplexes could be formed at the physiological pH of 7.0 in the presence of physiological concentrations (0.5-1 mM) of spermine. This stabilizing effect of polyamines was attributed to the changes in the overall charge density of the triplex DNA brought about by polyamine-DNA interactions. Initially, the triplex may bind with polyamines more strongly than the duplex because of a higher negative charge of the former, resulting in a shift of the equilibrium in favor of triplex formation.

Thomas and Thomas (48) questioned whether polyamine structure played a role in triplex DNA stabilization. To address this problem, they conducted Tm measurements of poly(dA)/2poly(dT) triplex in the presence of putrescine, spermidine, spermine, and homologs of putrescine and spermidine (Figs. 13 and 14). In the presence of polyamines, the absorbance vs temperature profile showed two transitions: Tm1, corresponding to triplex ^ duplex+single-stranded DNA, and Tm2, corresponding to duplex melting.

Fig. 13. Structural specificity effects of putrescine homologs on the melting temperature of triplex (Tmi) and duplex (Tm2) forms of DNA. The number of methylene groups (n) in putrescine homologs is plotted against the melting temperature at different diamine concentrations. (Reprinted with permission from ref. 48. © 1993 American Chemical Society.)

Fig. 13. Structural specificity effects of putrescine homologs on the melting temperature of triplex (Tmi) and duplex (Tm2) forms of DNA. The number of methylene groups (n) in putrescine homologs is plotted against the melting temperature at different diamine concentrations. (Reprinted with permission from ref. 48. © 1993 American Chemical Society.)

Fig. 14. Structural specificity effects of spermidine homologs on the melting temperature of triplex (Tml) and duplex (Tm2) forms of DNA. The number of methylene groups (n) in putrescine homologs is plotted against the melting temperature at different diamine concentrations. (Reprinted with permission from ref. 48. © 1993 American Chemical Society.)

Fig. 14. Structural specificity effects of spermidine homologs on the melting temperature of triplex (Tml) and duplex (Tm2) forms of DNA. The number of methylene groups (n) in putrescine homologs is plotted against the melting temperature at different diamine concentrations. (Reprinted with permission from ref. 48. © 1993 American Chemical Society.)

Figures 13 and 14 show the structural specificity of the various analogs, which vary in the number of methylene (-CH2-) groups, plotted against the Tm. In the presence of 0.5 mM putrescine, Tm1 and Tm2 were 44.8° and 71°C, respectively, in 10 mM Na cacody-late buffer. With 2.5 p,Mspermidine or 0.1 p,Mspermine, Tm1 values were 42.8°C and 54.4°C and Tm2 values were 65 and 82°C, respectively. These results showed that the ability of natural polyamines to stabilize the triplex DNA increased in the following order: spermine > spermidine > putrescine. In a series of putrescine homologs,

H2N(CH2)nNH2 (in which n = 2-6; n = 4 for putrescine), diaminopropane (n = 3) was the most effective stabilizer of triplex DNA. Among a series of triamines H2N(CH2)3-NH(CH2)nNH2 (in which n = 2-8; n = 4 for spermidine), spermidine was the most effective triplex stabilizing agent. Similar structural specificity was found with spermine homologs, H2N(CH2)3NH(CH2)nNH(CH2)3NH2 (in which n = 2-12; n = 4 for spermine). Interestingly, effects of these homologs on the poly(dA)/poly(dT) duplex were relatively insensitive to changes in the length of the methylene-bridging region. Differential effects of polyamine analogs in stabilizing triplex vs duplex DNA suggested their potential application in triplex DNA-based antigene therapeutics.

Effects of polyamines on the stabilization of triplex DNA formed from a purine motif triplex-forming oligonucleotide, 5'-TG3TG4TG4TG3T-3', and its target duplex probe, consisting of the oligonucleotides 5'-TCGAAG3AG4AG4AG3A-3' and 5'-TCGATC3TC4TC4TC3T-3', were studied in the presence of natural and synthetic polyamines (49). Electrophoretic mobility shift assay showed that 6is(ethyl) analogs of spermine and its higher analog, 4-4-4-4 were excellent stabilizers of triplex DNA (Fig. 15). In contrast, the non-Ms(ethyl) substituted parent polyamines aggregated the oligonucleotides in preference to triplex DNA stabilization. Temperature-dependent circular dichroism (CD) spectra of triplex DNA showed monophasic melting transition in the absence and presence of polyamines, suggesting duplex/triplex single-stranded DNA transition. These results indicate that structural modifications of polyamines are an effective strategy to develop triplex DNA-stabilizing ligands, with potential applications in antigene therapeutics. For example, diaminopropane stabilized triplex DNA and suppressed the c-myc oncogene expression in MCF-7 breast cancer cells at a level higher than that of controls in the absence of the diamine (50).

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