Ultrasensitive detection of DNA and protein markers in cancer cells.

Cancer cells differ from normal cells in various parameters, and these differences are caused by genomic mutations and consequential altered gene expression. The genetic and functional heterogeneity of tumor cells is a major challenge in cancer research, detection, and effective treatment. As such, the use of diagnostic methods is important to reveal this heterogeneity at the single-cell level. Droplet microfluidic devices are effective tools that provide exceptional sensitivity for analyzing single cells and molecules. In this review, we highlight two novel methods that employ droplet microfluidics for ultra-sensitive detection of nucleic acids and protein markers in cancer cells. We also discuss the future practical applications of these methods.

PNA openers and their applications for bacterial DNA diagnostics.

The unique ability of triplex-forming PNAs to invade the double helix has made it possible to develop a highly specific and sensitive approach for bacterial detection. The method uses short, about 20-bp-long, signature sequences presented as a single copy in the bacterial genome. Bacterial cells are fixed on slides and the PD-loop structure is assembled on the signature site with the help of PNA openers, which includes the circular probe. The sensitivity of the method is achieved via Rolling Circle Amplification (RCA) of the circular probe. The obtained amplicon is detected using short ssDNA decorator probes carrying fluorophores and via standard fluorescent microscopy.

Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.

We present an approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single-copy sensitivity and single-base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this technique by successfully detecting six single-copy target sites on human mitochondrial and autosomal DNA. We also demonstrate the high selectivity of this method by detecting X- and Y-specific sequences on human sex chromosomes and by simultaneously detecting three sequence-specific target sites. Finally, we discriminate two target sites that differ by 2 nt. The PNA-RCA-FISH approach is a distinctive in situ hybridization method capable of multitarget visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.

Target DNA detection and quantitation on a single cell with single base resolution.

In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.

Fluorescence-based detection of short DNA sequences under non-denaturing conditions.

The ability of peptide nucleic acid (PNA) to open up duplex DNA in a highly sequence-specific manner makes it possible to detect short DNA sequences on the background of or within genomic DNA under non-denaturing conditions. To do so, chosen marker sites in double-stranded DNA are locally opened by a pair of PNA openers, thus transforming one strand within the target region (20-30 bp) into the single-stranded form. Onto this accessible DNA sequence a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). Both homogeneous and heterogeneous assay formats are investigated, as are different formats for fluorescence-based amplicon detection. Our recent data with immobilized analytes suggest that marker sequences in plasmid and bacterial chromosomal DNA can be successfully detected.

End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes.

Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine-homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA-DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

Sequence-universal recognition of duplex DNA by oligonucleotides via pseudocomplementarity and helix invasion.

The well-known Watson-Crick complementarity rules, which were discovered 50 years ago, elegantly direct the specific pairing of two DNA single strands. On the contrary, once formed, the double-stranded (ds) DNA lacks such a simple and sequence-universal recognition principle, since most of the characteristic chemical groups of nucleobases are now buried deep inside the double helix, the major DNA form. We report a promising versatile approach for highly selective recognition of designated sites within dsDNA featuring considerable practical potential for a variety of molecular-biological, biotechnological, gene-therapeutic, and diagnostic applications. It may also have implications for prebiotic evolution of genetic machinery at the primordial stages of the origin of life. Our design synergistically employs the robust helix-invasion ability of recently developed DNA mimics and analogs, pseudocomplementary peptide nucleic acids and pseudocomplementary oligonucleotides, thus enabling the sequence-unrestricted recognition of chosen DNA duplexes by nucleobase oligomers. Using this basically general approach, we selectively tagged a unique mixed-base site on the target dsDNA fragment with streptavidin and/or multiply labeled this site with fluorophores via the primer-extension reaction.

Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro.

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ø29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A(3) (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.