Effects of chain length modification and bis(ethyl) substitution of spermine analogs on purine-purine-pyrimidine triplex DNA stabilization, aggregation, and conformational transitions.
The natural polyamines--putrescine, spermidine, and spermine--are known to stabilize pyrimidine-purine-pyrimidine and purine-purine-pyrimidine triplex DNA formation. We studied the ability of two tetramine and two pentamine analogs of spermine and their bis(ethyl) derivatives to stabilize triplex DNA formation between 5'-TG3TG4TG4TG3T-3' and its target duplex probe, consisting of the oligonucleotides 5'-TCGAAG3AG4AG4AG3A-3' and 5'-TCGATC3TC4TC4TC3T-3'. We used electrophoretic mobility shift assay (EMSA), melting temperature (Tm) measurements, and circular dichroism (CD) spectroscopy to evaluate the effects of these novel polyamine analogs on triplex DNA stability, dissociation constants, aggregation, and conformation. In general, pentamines were more efficacious than tetramines in stabilizing triplex DNA, although most of the polyamines with pendant free amino groups caused DNA aggregation below 50% conversion to triplex DNA. Ethyl substitution of these pendant amino groups lowered their efficacy approximately 2-fold in stabilizing triplex DNA; however, this effect was more than compensated for by the lack of DNA aggregation in the presence of bis(ethyl)polyamines. A concentration-dependent increase in the Tm of triplex DNA was observed in the presence of polyamines. CD spectral measurements showed distinct differences in the conformation of triplex DNA stabilized in the presence of polyamines compared to the CD spectra of the oligonucleotides alone. Temperature-dependent CD spectra of triplex DNA showed monophasic melting in the absence and presence of polyamines, suggesting duplex/triplex --> single-stranded DNA transition. These results indicate that structural modifications of polyamines is an effective strategy to develop triplex DNA-stabilizing ligands, with potential applications in antigene therapeutics.
Musso
M
M
Department of Tumor Biology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Thomas
T
T
Shirahata
A
A
Sigal
L H
LH
Van Dyke
M W
MW
Thomas
T J
TJ
D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D013096:Spermine / Q000031:analogs & derivatives*; D013696:Temperature
C051802:triplex DNA; D013096:Spermine; D004247:DNA