Pseudocomplementary PNAs containing diaminopurine⅐thiouracil base pairs have been prepared and are shown to bind with high specificity and efficiency to complementary targets in doublestranded DNA by a mechanism termed ''double duplex invasion'' in which the duplex is unwound and both DNA strands are targeted simultaneously, each by one of the two pseudocomplementary peptide nucleic acids (PNAs). On the basis of our results we predict that (for decameric targets) more than 80% of all sequences can be targeted by straightforward Watson-Crick base pairing by using this approach in its present form. Targeting of pseudocomplementary PNAs to the promoter of the T7 phage RNA polymerase effectively inhibits transcription initiation. These results have important implications in the development of gene therapeutic agents as well as for genetic diagnostic and molecular biology applications.
In clinical routine pathology today, detection of protein in intact formalin-fixed, paraffin-embedded tissue is limited to immunohistochemistry, which is semi-quantitative. This study presents a new and reliable quantitative immunohistochemistry method, qIHC, based on a novel amplification system that enables quantification of protein directly in formalin-fixed, paraffin-embedded tissue by counting of dots. The qIHC technology can be combined with standard immunohistochemistry, and assessed using standard bright-field microscopy or image analysis. The objective was to study analytical performance of the qIHC method. qIHC was tested under requirements for an analytical quantitative test, and compared with ELISA and flow cytometry for quantitative protein measurements. Human epidermal growth factor receptor 2 (HER2) protein expression was measured in five different cell lines with HER2 expression from undetectable with immunohistochemistry to strong positive staining (IHC 3+). Repeatability, reproducibility, robustness, linearity, dynamic range, sensitivity, and quantification limits were evaluated. Reproducibility and robustness were assessed in a setup to resemble daily work in a laboratory using a commercial immunohistochemistry platform. In addition, qIHC was correlated to standard HER2 immunohistochemistry in 44 breast cancer specimens. For all evaluated parameters, qIHC performance was either comparable or better than the reference methods. Furthermore, qIHC has a lower limit of detection than both immunohistochemistry and the ELISA reference method, and demonstrated ability to measure HER2 accurately and precise within a large dynamic range. In conclusion, the results show that qIHC provides a sensitive, quantitative, accurate, and robust assay for measurement of protein expression in formalin-fixed, paraffin-embedded cell lines, and tissue.
Fluorescein ethyl ester, I, was used to prepare the fluorescent mixed ester/ether 6-O-(carboxymethyl)-fluorescein ethyl ester, III. Conjugation of III to the epsilon-amino group of alpha-N-Boc-L-lysine, via the N-hydroxysuccinimde ester, IV, gave the Boc-protected fluorescein-conjugated lysine monomer V. Removal of the Boc group, followed by reaction with Fmoc chloride, gave the Fmoc-protected monomer, VI (Figure 1). These Boc- and Fmoc-protected fluorescein-conjugated lysines were readily incorporated into peptides and PNA oligomers during solid phase synthesis to give fluorescent products. Mass spectroscopy and UV studies showed that the fluorophore remains unchanged during solid phase synthesis. In contrast to fluorescein, the photophysical properties of these derivatives are pH independent from pH 3 to 8, with a molar absorption coefficient, epsilon max 456, of 2.9 x 10(4) M-1 cm-1 and fluorescence quantum yield, phi f, of 0.18.
Novel reporters have been synthesized with extended hydrophilic linkers that in combination with polymerizing cross-linkers result in very efficient reporter deposition. By utilizing antibodies to stain HER2 proteins in a cell line model it is demonstrated that the method is highly specific and sensitive with virtually no background. The detection of HER2 proteins in tissue was used to visualize individual antigens as small dots visible in a microscope. Image analysis-assisted counting of fluorescent or colored dots allowed assessment of relative protein levels in tissue. Taken together, we have developed novel reporters that improve the CARD method allowing highly sensitive in situ detection of proteins in tissue. Our findings suggest that in situ protein quantification in biological samples can be performed by object recognition and enumeration of dots, rather than intensity-based fluorescent or colorimetric assays.
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