Abstract: There are many situations where oligonucleotides that efficiently bind a target DNA or RNA are desired. These oligonucleotides can be used for a variety of purposes, including antisense, diagnostics, and array generation. While researchers have worked for many years to identify algorithms and methods for predicting the oligonucleotides that will bind the target with the highest efficiency, better prediction methods are needed. Disclosed are methods, articles, machines, and compositions that aid in identifying oligonucleotides and sets of oligonucleotides that will efficiently bind a target nucleic acid molecule. Also disclosed are optimized sets of oligonucleotides that bind HIV-1 genomic RNA or DNA, such as the GAG RNA, and methods of using them.

Abstract: A tissue culture assay for measuring drug-induced recoding in regulating cellular polyamine levels is described. A DNA construct containing the renilla luciferase gene separated by sort cloning site from the firefly luciferase gene, both under the control of a single upstream SV40 promoter is provided. The cloning site contains the portion of antizyme gene known to contain the mRNA signals for polyamine stimulated frameshifting with the downstream firefly gene in the +1 position relative to the upstream renilla gene. A control construct is also produced with the genes in the same reading frame. Frameshifting efficiencies can be determined by comparing the ratio or firefly to renilla luciferase activity in parallel cell cultures.

Abstract: An artificial neural network system for analyzing sequence motif content for prediction of antisense oligonucleotide-target activity is disclosed. The system was developed for high specificity predictions, with cross-validation used to rigorously test against the database that was used in the development of the system. The system is able to choose effective oligonucleotides leading to >75% reduction in RNA target expression with >55% accuracy. This is in contrast to <10% success rate for trial-and-error oligonucleotide selection. Thus, the program provides a five-fold reduction in the number of oligonucleotides to be screened in vivo to find effective targets.

Abstract: Assays for screening small-molecule compounds for their ability to induce translational readthrough of stop codons are disclosed. The assays utilize a dual enzymatic reporter plasmid system, wherein one reporter acts as an internal standard and the second reporter measures the translational recoding event induced by the small-molecules. The genetic sequence mutations of interest are placed on the plasmid between the two reporter genes and the plasmids are transfected into tissue culture cells. The cells are then grown in the presence of varying amounts of small-molecule compounds and the induction of translational readthrough is measured.

Abstract: A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus.

Abstract: Plasmids and methods of use for assaying translational recoding are disclosed. The plasmids contain a constitutively expressed renilla (Renilla reniformis; sea pansy) luciferase gene, a polylinker for insertion of a selected DNA segment, and an out-of-frame firefly luciferase gene. Recoding is determined by monitoring luminescence of the firefly luciferase normalized to the luminescence of the renilla luciferase.

Abstract: A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus.