ABSTRACT
Over the past two decades, synthetic oligonucleotides have become a prime area of focus in biopharmaceutical drug discovery and development pipelines for the potential treatment of a broad range of disease indications including cancer, diabetes, viral infections, cardiovascular disease, as well as many inªammatory and degenerative disorders. Numerous oligonucleotide-based therapeutics are actively being investigated in both preclinical and clinical settings, with a ‰rst generation phosphorothioate antisense oligonucleotide (ASO), Vitravene™, approved by the Food and Drug Administration (FDA) in 1998 for the antiviral treatment of cytomegalovirus retinitis infection.1 Since that time, there has been signi‰cant progress in the oligonucleotide ‰eld with the development of different classes of oligonucleotide-based therapeutic candidates, encompassing a number of different chemical modi‰cations, drug delivery modalities and proposed mechanisms of action. Investigational oligonucleotide therapeutics, which have and continue to show great promise in the drug development pipeline, include the antisense oligonucleotides (ASOs),2 small interfering RNAs (siRNAs),3-5 immunomodulatory oligonucleotides (IMOs),6 and aptamers.7 Other lesser investigated classes include decoy oligonucleotides8,9 and ribozymes/DNAzymes.10,11 The potential applications of many of these oligonucleotide classes as viable drugs in the treatment of human diseases remains
8.1 Introduction and Overview ...................................................................................................265 8.2 Hybridization Techniques and Instrumentation-General Considerations .......................... 267
8.2.1 Ligation-Dependent Hybridization ELISA ............................................................... 270 8.2.2 Nuclease-Dependent Hybridization ELISA .............................................................. 273 8.2.3 Dual-Probe Hybridization ELISA ............................................................................ 275 8.2.4 Competitive Hybridization ELISA ........................................................................... 275
8.3 Experimental Setup .............................................................................................................. 276 8.3.1 Bioanalysis Using a Nonextraction Procedure ......................................................... 276 8.3.2 Bioanalysis Using an Extraction Procedure ............................................................. 277 8.3.3 Method Development and Validation ....................................................................... 277
8.4 Conclusions and Summary ................................................................................................... 279 References ......................................................................................................................................280
in its infancy. Nonetheless, remarkable progress has been made to improve upon the stability, ef‰- cacy, safety, potency, and delivery of oligonucleotide therapeutics, much of which has been gained from the exceptional knowledge acquired through second-generation antisense oligonucleotide research programs.
