In situ synthesis of oligonucleotide arrays by using surface tension.

This work describes the in situ synthesis of oligonucleotide arrays on glass surfaces. These arrays are composed of features defined and separated by differential surface tension (surface tension arrays). Specifically, photolithographic methods were used to create a series of spatially addressable, circular features containing an amino-terminated organosilane coupled to the glass through a siloxane linkage. Each feature is bounded by a perfluorosilanated surface. The differences in surface energies between the features and surrounding zones allow for chemical reactions to be readily localized within a defined site. The aminosilanation process was analyzed using contact angle, X-ray photoelectron spectroscopy (XPS), and time-of-flight/secondary ion mass spectroscopy (TOF-SIMS). The efficiency of phosphoramidite-based oligonucleotide synthesis on these surface tension arrays was measured by two methods. One method, termed step-yields-by-hybridization, indicates an average synthesis efficiency for all four (A,G,C,T) bases of 99.9 +/- 1.1%. Step yields measured for the individual amidite bases showed efficiencies of 98.8% (dT), 98.0% (dA), 97.0% (dC), and 97.6% (dG). The second method for determining the amidite coupling efficiencies was by capillary electrophoresis (CE) analysis. Homopolymers of dT (40- and 60mer), dA (40mer), and dC (40mer) were synthesized on an NH(4)OH labile linkage. After cleavage, the products were analyzed by CE. Synthesis efficiencies were calculated by comparison of the full-length product peak with the failure peaks. The calculated coupling efficiencies were 98.8% (dT), 96.8% (dA), and 96.7% (dC).

Effects of excitatory amino acids on the hypothalamic-pituitary-adrenal axis of the neonatal rat.

Most stimuli that elicit a response by the hypothalamic-pituitary-adrenal (HPA) axis of adult rats fail to do so in infant rats aged 4-14 postnatal days (pnd). This interval is termed the stress hyporesponsive period (SHRP). The present study examined the development of the HPA response to the excitatory amino acids (EAAs), N-methyl-D-aspartic acid (NMDA) and kainic acid (KA), at 3 ages (i.e., pnd 6, 12, 18) during or immediately after the SHRP. Results indicate that intraperitoneal (i.p.) administration of 2.5 mg/kg KA or 5 mg/kg NMDA is capable of inducing age- and time-dependent elevations of ACTH and CORT, with KA being the more potent of the two EAAs. In contrast to other stimuli which are capable of eliciting an HPA response during the SHRP, NMDA and KA appear to possess more potent effects at earlier ages. Administration of lower doses of these EAAs did not elicit an HPA response. Pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 0.69 mg/kg i.p.), a KA receptor-specific antagonist, attenuated the effects of KA. These results suggest that KA exerts its effects via the KA receptor and that this receptor appears to be mature at both pnd 12 and 18. In contrast, pretreatment with D,L-2-amino-5 phosphonovaleric acid (APV; 7.5 mg/kg i.p.), an NMDA receptor-specific antagonist, was only effective at pnd 18 suggesting that the NMDA receptor is not yet mature at pnd 12. Finally, EAAs induce age- and time-dependent behavioral modifications (i.e., hindpaw scratching and hyperlocomotion). These effects, however, appear to only contribute to, but not cause, the endocrine responses.