Multiplexed and Reiterative Detection of Protein Markers inCells using Dynamic Nucleic Acid Complexes

摘要

The diagnosis, staging and clinical management of cancer and other diseases isbecoming increasingly reliant upon the identification and quantification of molecularmarkers as well their spatial distribution in histological samples. Yet, due to spectraloverlap of dyes and the inability to remove probes without affecting marker integrity,immunohistological methods are limited by the number of markers that can be examinedon a single specimen resulting in loss of information that could be vital to diagnosis ortreatment.This dissertation describes the development and characterization of an erasablemulti-color imaging technology capable of detecting large numbers of molecular markerson a single biological sample. The system consists of (1) 'targets', which are single orpartially hybridized DNA strands conjugated to a protein of interest for biomarkerrecognition in cells, and (2) multi-strand, fluorophore-containing DNA 'probecomplexes' that react with the DNA portion of the target in a sequence dependent fashionto create fluorescent reporting complexes. The addition of a quencher-bearing ssDNAdisplaces the target's DNA strand to effectively remove the dye from the marker so thatthe sample can be re-imaged for other markers with minimal interference from prioriiirounds of labeling. Orthogonal DNA sequences and spectrally-separated dyes can be usedto create multiple, unique target/probe pairs that associate specifically and can be imagedin parallel.The overall utility of this technology depends on high specificity of targets torespective probe complexes, highly efficient labeling and erasing to ensure thatfluorescent signals can be used to fully quantify target abundance without the interferenceof signals from previous rounds of labeling, and short reaction times to allow for multiplerounds of processing on the same sample without loss of integrity. Based on the abovecriteria, three classes of probes were designed and their structure-function relationshipselucidated to determine the contributions of complex size, free energy differencesbetween intermediate states, and strand displacement on labeling and erasing kinetics andefficiencies on cells.A comparison of the kinetics of the labeling and erasing reactions for the threedifferent constructs showed that reaction efficiencies depend less on calculated net freeenergy change than on the engineered state of the complex during the stranddisplacement reaction (i.e., the type of strand displacement reaction it participates in).This new paradigm in probe design allowed the system to meet its design goals,potentially increasing the diagnostic power of individual histological specimens andopening the door to more sophisticated analyses of cell phenotype and its functionalrelationship to disease.