Binding between an amine cationic surfactant and DNA and surface properties of the resultant aggregates.

Binding between cetyltrimethylammonium bromide, a cationic surfactant, and a variety of lengths of single stranded DNA was measured using fluorescence polarization and a simple cooperative model was used to obtain dissociation constants on the order of 1 × 10-5 for the aggregates that formed. Aggregation depended on strand length where strands much shorter than 40 nucleotides (for example strands of 24-nucleotides) were too short to form the same size aggregates. Other factors such as salt concentration and temperature also affected aggregate formation: increasing either the salt concentration or performing binding at the highest temperature studied (60 °C) made it more difficult for aggregates to form. Both heating and dilution of aggregates caused the anisotropy signal to decrease, which suggested that the complexes fell apart under these conditions. Force spectroscopy of aggregate surfaces showed that both electrostatic and hydrophobic adhesive forces were present between aggregates and derivatized AFM tips. These findings can be used to better understand the stability of cationic surfactant-DNA aggregates and may provide guidance for lipid nanoparticle design used in vaccine development and therapeutics.

Varying Susceptibility of the Female Mammary Gland to In Utero Windows of BPA Exposure.

In utero exposure to the endocrine disrupting compound bisphenol A (BPA) is known to disrupt mammary gland development and increase tumor susceptibility in rodents. It is unclear whether different periods of in utero development might be more susceptible to BPA exposure. We exposed pregnant CD-1 mice to BPA at different times during gestation that correspond to specific milestones of in utero mammary gland development. The mammary glands of early-life and adult female mice, exposed in utero to BPA, were morphologically and molecularly (estrogen receptor-α and Ki67) evaluated for developmental abnormalities. We found that BPA treatment occurring before mammary bud invasion into the mesenchyme [embryonic day (E)12.5] incompletely resulted in the measured phenotypes of mammary gland defects. Exposing mice up to the point at which the epithelium extends into the precursor fat pad (E16.5) resulted in a nearly complete BPA phenotype and exposure during epithelial extension (E15.5 to E18.5) resulted in a partial phenotype. Furthermore, the relative differences in phenotypes between exposure windows highlight the substantial correlations between early-life molecular changes (estrogen receptor-α and Ki67) in the stroma and the epithelial elongation defects in mammary development. These data further implicate BPA action in the stroma as a critical mediator of epithelial phenotypes.

Effects of locked nucleic acid substitutions on the stability of oligonucleotide hairpins.

An understanding of the stability of nucleic acid folding is critical for applications involving RNA viruses, small molecule-RNA binding, and therapeutics, for example. To explore factors that affect this stability, hairpins made from oligonucleotides containing both a GAAA tetraloop and three to five complements in the stem have been used as models where locked nucleic acids (LNAs) have been substituted into the sequence. UV spectroscopy was used to obtain melting curves in 20% by volume formamide, and the enthalpies and entropies of melting were determined. Although LNA substitutions typically increase the stability of a hybrid, we have found a decrease in stability for DNA and RNA GAAA hairpins when LNA is substituted into the loop. Tetraloops synthesized from natural bases show higher enthalpies and entropies of melting compared to the LNA substituted sequences indicating that LNA substitutions can destabilize a hairpin but stabilize the corresponding double stranded structure.

Increase in hybridization rates with oligodeoxyribonucleotides containing locked nucleic acids.

Locked nucleic acids (LNAs) incorporated into either stable single stranded oligonucleotides containing tetraloops or their complements have been found to increase second order hybridization rate constants by an order of magnitude compared to the all-DNA hybridization rate constants. Model sequences composed of 20 bases in length that can form hairpins due to a stable GAAA tetraloop were used where LNAs were substituted for the nucleotides in the loop, stem, or end regions of the strand and in the complementary strand. Substitution of the LNAs to the loop predictably raised the melting temperatures of the duplex however, the hybridization rates between the tetraloop and the complementary sequence also increased. In contrast, when LNAs were substituted in the stem, the hybridization rate decreased implying the formation of a more stable hairpin. Substitution of LNAs into the end region of the sequence had little effect on the hybridization rate constants although melting temperatures still showed a predictable increase. Rates also increased when LNAs were substituted into complementary strands of DNA tetraloops. The increase in hybridization rate constant is being attributed to changes in the structure of the stable single strands.

Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres.

Hybridization kinetics of DNA sequences with known secondary structures and random sequences designed with similar melting temperatures were studied in solution and when one strand was bound to 5 mum silica microspheres. The rates of hybridization followed second-order kinetics and were measured spectrophotometrically in solution and fluorometrically in the solid phase. In solution, the rate constants for the model sequences varied by almost two orders of magnitude, with a decrease in the rate constant with increasing amounts of secondary structure in the target sequence. The random sequences also showed over an order of magnitude difference in the rate constant. In contrast, the hybridization experiments in the solid phase with the same model sequences showed almost no change in the rate constant. Solid phase rate constants were approximately three orders of magnitude lower compared with the solution phase constants for sequences with little or no single-stranded structure. Sequences with a known secondary structure yielded solution phase rate constants as low as 3 x 10(3) M(-1) s(-1) with solid phase rate constants for the same sequences measured at 2.5 x 10(2) M(-1) s(-1). The results from these experiments indicate that (i) solid phase hybridization occurs three orders of magnitude slower than solution phase, (ii) trends observed in structure-dependent kinetics of solution phase hybridization may not be applicable to solid phase hybridization and (iii) model probes with known secondary structure decrease reaction rates; however, even random sequences with no known internal single-stranded structure can yield a broad range of reaction rates.