Method for detecting binding efficiencies of synthetic oligonucleotides: Targeting bacteria and insects

Authors: Hunter, Wayne; METZ, JACKIE - University Of Florida; ALTMAN, SIDNEY - Yale University; AISHWARYA, VEENU - Aum Lifetech; PELZ-STELINSKI, KIRSEN - University Of Florida

Submitted to: Arthropod Management Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 5/3/2017
Publication Date: 6/8/2017
Citation: Hunter, W.B., Metz, J.L., Altman, S., Aishwarya, V., Pelz-Stelinski, K. 2017. Method for detecting binding efficiencies of synthetic oligonucleotides: Targeting bacteria and insects. In: Proceedings of 2017 10th Arthropod Genomics Symposium, June 8-11, 2017, Notre Dame, Indiana. p. 45

Interpretive Summary: An improved method was developed to quantify binding efficiency of two antisense oligonucleotide (ASO), molecules which bind to and degrade bacterial or insect nucleotides. These ASO bind in a sequence specific manner and can be used to reduce hard to manage plant and animal pathogens. The gene-based binding biotechnology is emerging as a potential treatment for drug resistant bacteria. The new method measures how strong an ASO will bind to the specific nucleotide. Once bound the cell's natural degrading enzymes detect the location where bound and will cut the nucleotide which blocks formation of the specific protein. The method can be used as a prescreening protocol ASO molecules to validate binding efficiency prior to testing in plants or insects thus reducing costs and saving time. Similar methods apply across technologies like Ribonucleic acid interference, Ribonucleic acid interference (RNAi). Supported in-part: National Institute of Food and Agriculture (NIFA), USDA, Citrus Greening award #2015-70016-23028; and NIFA, USDA, award #2015-10479.

Technical Abstract: Expanding applications of gene-based targeting biotechnology in functional genomics and the treatment of plants, animals, and microbes has synergized the need for new methods to measure binding efficiencies of these products to their genetic targets. The adaptation and innovative use of Cell–Penetrating-Peptide Morpholino, and antisense oligonucleotides depend on visual bound fluorophores and confocal microscopy, or intensive chemical Gas-Chromatography-Mass Spectrometry analyses for confirmation of delivery. Confirmation of the presence of labeled molecules can be detected using fluorescent plate readers which provides a more rapid and cost effective analyses. However, neither confocal or plate reader, addresses the binding efficiency, binding competition, or detection of unlabeled oligonucleotides. Presented is a method using primers which flank or reside on the binding target as a means to quantify binding preference across known targets. The method can also be used in qualitative Polymerase Chain Reaction (qPCR), analyses to quantify bound to unbound oligonucleotides. As a prescreening method to validate binding efficiency, cost savings and reduced time are possible. Similar methods apply across technologies like Ribonucleic acid interference (RNAi). Supported in-part: National Institute Food and Agriculture (NIFA), USDA, Citrus Greening award #2015-70016-23028; and NIFA, USDA, award #2015-10479.