Exploring protein conformation-binding relationships and antibody glyco-profiles using a nanopore transduction detector

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The relation between protein structure and function is well known and minor changes in protein folding or isoform variants, or surface modifications such as glycosylations, can impact that protein function. To complicate matters further, many proteins are inherently dynamic, so their structure-function relationship can give rise to dynamic functionality, with selection sometimes favoring very dynamic proteins. What is needed is a means to track protein conformation and its role in protein function, binding in particular, and this suggests we need a means to track the conformational state of a single protein. A method for using a nanopore transduction detector (NTD) is proposed for such an application. NTD transducers are molecules that serve to transduce the conformational or binding state of a molecule of interest into different channel current modulations, where the molecule of interest is tethered to a nanopore channel modulator. In previous work, using inexpensive biomolecular components, such as DNA hairpin channel-modulators, antibodies, and immuno-PCR linkages to antibodies, experiments were done to analyze individual antibodies and DNA molecules. Three complications were indicated before a general-use NTD platform could be established: (1) the convenient DNA-based modulators were often too short-lived for the binding study of interest; (2) the transducer’s bound state often didn’t modulate; and (3) the binding target often had a pI that didn’t favor being drawn to the channel-tethered study molecule. In recent work NTD operation has been demonstrated for a wide range of pH, chaotrope concentration, and in the presence of interference agents, such that problem (3) is solved. While in the latest results shown here the other problems are solved as well: very long-lived channel modulators are demonstrated using locked nucleic acid (LNA) nucleosides; and induced modulations are demonstrated by engineering transducers to receive laser-tweezer impulses by means of a linked magnetic bead (another commoditized component). A general-use NTD platform is thereby possible using an alpha-hemolysin nanopore detector and performing the critical transducer engineering with readily available, inexpensive (commoditized), biomolecular components.