The diagnosis, staging and clinical management of cancer and other diseases is
becoming increasingly reliant upon the identification and quantification of molecular
markers as well their spatial distribution in histological samples. Yet, due to spectral
overlap of dyes and the inability to remove probes without affecting marker integrity,
immunohistological methods are limited by the number of markers that can be examined
on a single specimen resulting in loss of information that could be vital to diagnosis or
treatment.
This dissertation describes the development and characterization of an erasable
multi-color imaging technology capable of detecting large numbers of molecular markers
on a single biological sample. The system consists of (1) 'targets', which are single or
partially hybridized DNA strands conjugated to a protein of interest for biomarker
recognition in cells, and (2) multi-strand, fluorophore-containing DNA 'probe
complexes' that react with the DNA portion of the target in a sequence dependent fashion
to create fluorescent reporting complexes. The addition of a quencher-bearing ssDNA
displaces the target's DNA strand to effectively remove the dye from the marker so that
the sample can be re-imaged for other markers with minimal interference from prior
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rounds of labeling. Orthogonal DNA sequences and spectrally-separated dyes can be used
to create multiple, unique target/probe pairs that associate specifically and can be imaged
in parallel.
The overall utility of this technology depends on high specificity of targets to
respective probe complexes, highly efficient labeling and erasing to ensure that
fluorescent signals can be used to fully quantify target abundance without the interference
of signals from previous rounds of labeling, and short reaction times to allow for multiple
rounds of processing on the same sample without loss of integrity. Based on the above
criteria, three classes of probes were designed and their structure-function relationships
elucidated to determine the contributions of complex size, free energy differences
between intermediate states, and strand displacement on labeling and erasing kinetics and
efficiencies on cells.
A comparison of the kinetics of the labeling and erasing reactions for the three
different constructs showed that reaction efficiencies depend less on calculated net free
energy change than on the engineered state of the complex during the strand
displacement 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 and
opening the door to more sophisticated analyses of cell phenotype and its functional
relationship to disease