Abstract
Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.
Highlights
In this work we describe the application of DNAzyme peroxidase joined to aptamers as a standardized tool to stain the PTEN tumor suppressor protein in murine brain, human colon, and endometrial hyperplasia and cancer specimens
We used histological sections prepared from mouse cerebellum for the following reasons: (i) The mouse cerebellum is an excellent model for PTEN studies because the subcellular localization of this protein can be observed in Purkinje cells, the largest neurons in the brain, as we previously demonstrated by the rabbit antiserum and aptamers that are used in this study [27]. (ii) The aptamer PTENz7-87 targets the first 7 amino acids present in a region of a 14 amino acids sequence conserved in both murine and human PTEN protein. (iii) The aptamer PTENz14-87 targets the last seven amino acids of this conserved sequence
We showed that a shorter dual PTEN aptamer gave identical results when used in histological sections prepared from human endometrial and colon tissues (Figure 4A–F)
Summary
Aptamers could be any natural, synthetic, or modified polymer, most common nucleic acid and peptides that bind to a specific target molecule. Aptamers are highly resistant to degradation and can be chemically synthesized at a relatively low cost with high reproducibility. They can be customized to incorporate chemical modifications and can be joined with affinity tags or labels. The same year, our group described the target switching method to produce a synthetic oligonucleotide capable of recognizing protein phosphatase 2A (PP2A)
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