Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.

We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequence of cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monitored by enhanced fluorescence of the beacon, due to the weakening of fluorescence energy transfer, upon its binding to the target duplex. Electrophoretic mobility shift assay confirmed triplex DNA formation by these oligonucleotides. In low salt buffer (10 mM Na(+)), triplex DNA formation was not observed in the absence of a ligand such as spermine. At room temperature (22 degrees C), the equilibrium association constant (K(a)) calculated in the presence of 1 microM spermine and 10 mM Na(+) was 3.2 x 10(8) M(-1). The K(a) value was 1.0 x 10(9) M(-1) in the presence of 150 mM Na(+), and it increased by 10-fold by the addition of 1 mM spermine. Delta H, Delta S, and Delta G of triplex DNA formation, calculated from the temperature dependence of K(a) in the range of 20--45 degrees C, were -35.9 kcal/mol, -77 cal/(mol.K), and -13 kcal/mol, respectively, in the presence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(mol.K), and -13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyamines exerted different degrees of triplex DNA stabilization, as determined by binding constant measurements. Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant, whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplex and polyamines, the molecular beacon dissociated with a melting temperature of 67 degrees C. Thermodynamic parameters of beacon melting were calculated from the melting curve, and the Delta H, Delta S, and Delta G values were 37.8 kcal/mol, 112 cal/(mol.K), and 4.4 kcal/mol, respectively. These results demonstrate that molecular beacons can be used for the direct determination of the equilibrium association constants and thermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.

We synthesized analogues of spermine and studied the effects of chemical structure, ionic strength, and temperature on lambda-DNA nanoparticle formation. Effective concentration of polyamines for DNA condensation (EC50) was lowest for hexamines (0.2 microM) and highest for spermine (tetramine, 4.2 microM). The EC50 value increased with [Na+]. Dynamic light scattering showed nanoparticles with hydrodynamic radii (R(h)) of 40-50 nm. Effect of temperature on R(h) was measured between 20 and 70 degrees C. For spermine, R(h) remained relatively stable until 50 degrees C and increased significantly at >60 degrees C. In contrast, the hexa- and penta-valent analogues exhibited a gradual increase in R(h) between 20 and 70 degrees C. The nanoparticles were mainly toroidal, as revealed by electron microscopy (EM). EM studies showed changes in morphology and size of condensed structures with an increase in temperature. A possible mechanism for the differential effects of temperature on DNA nanoparticles might involve different modes of DNA-polyamine interactions.