Ultrasensitive detection of DNA and protein markers in cancer cells

PNA-RCA method for detection of short DNA target sites at the single-cell level. (A) (I) PNA openers specifically bind to two closely located homopurine DNA sites, which are separated by several random purine-pyrimidine bases, and locally open the double-stranded DNA; (II) The opened region serves as a target for hybridization and ligation of an oligonucleotide probe to form a PD-loop; (III) The small circle on duplex DNA serves as a template for isothermal RCA reaction, which yields a long, single-stranded amplicon that contains thousands of copies of the target sequence. For the fluorescence-based detection, fluorophore-tagged decorator probes are hybridized to the RCA product. (B) A map of viral genomic locus used for viral identification. (C) Target sites in the EBV genomic DNA used as genetic biomarkers. Sequences targeted by PNAs are shown in red. EBNA-3(G)-EBV type 1 signature sites within the EBNA-3 gene differ by a single nucleotide (SNP) from EBNA-3(T)-EBV type 2 in the PNA binding sequence. Mismatch is shown in black. (D) Multiple fluorescent spots were detected by fluorescent microscopy in BC-1 cells (EBV-positive) upon application of the probe corresponding to the LMP-1 gene encoding major transforming protein of EBV. The fluorescent signals were acquired separately using two filter sets, namely, DAPI for DNA and Cy3 for labeled RCA product; (E-G) For quantitation of oncoviral DNA target sites within BC-1 cells, fluorescence intensities were recorded for the single-copy genes LMP-1 and EBNA-3 and for multiple copies of the EBNA-2 IR target sites. Each droplet contains a single cell. Representative droplet images: (E) DIC; (F) CY3; (G) The fluorescence intensities from these images are plotted as 3D surface graphs using ImageJ (top panel, the color code at the right indicates fluorescence intensities).