Cationic Conjugated Polymers for Optical Detection of DNA Methylation, Lesions, and Single Nucleotide Polymorphisms

Abstract

Simple, rapid, and sensitive technologies to detect nucleic acid modifications have important applications in genetic analysis, clinical diagnosis, and molecular biology. Because genetic modifications such as single nucleotide polymorphisms (SNP), DNA methylation, and other lesions can serve as hallmarks of human disease, interest in such methods has increased in recent years. This Account describes a new strategy for the optical detection of these DNA targets using cationic conjugated polymers (CCPs). Because of their unique signal amplification properties, researchers have extensively investigated conjugated polymers as optical transducers in highly sensitive biosensors. Recently, we have shown that cationic polyfluorene can detect SNPs within the DNA of clinical samples. When we incorporated deoxyguanosine triphosphate (dGTP-Fl) into the DNA chain at an SNP site where the target/probe pair is complementary, we observed higher fluorescence resonance energy transfer (FRET) efficiency between cationic polyfluorene and fluorescein label on the dGTP. By monitoring the change in emission intensity of cationic polyfluorene or fluorescein, we identified the homozygous or heterozygous SNP. The high sensitivity of this assay results from the 10-fold enhancement of fluorescein emission intensity by the FRET from polyfluorene. This method can detect allele frequencies (the proportion of all copies of a gene that is made up of a particular gene variant) as low as 2%. Using this novel method, we clearly discriminated among the SNP genotypes of 76 individuals of Chinese ancestry. Improving on this initial system, we designed a method for multicolor and one-tube SNP genotyping assays based on cationic polyfluorene using fluorescein-labeled deoxyuridine triphosphate (dUTP-Fl) and Cy3-labeled deoxycytidine triphosphate (dCTP-Cy3) in extension reactions. We also developed a one-step method for direct detection of SNP genotypes from genomic DNA by combining allele-specific PCR with CCPs. In 2008, we developed a new method for DNA methylation detection based on single base extension reaction and CCPs. Treatment of DNA with bisulfite followed by PCR amplification converts unmethylated DNA into a C/T polymorphism, which allows us to characterize the methylation status of the target DNA. Furthermore, we used CCPs to detect DNA lesions caused by ultraviolet light irradiation for the first time. By monitoring the color change of cationic polythiophene before and after DNA cleavage, we also detected oxidative damage to DNA by hydroxyl radical. These CCP-based new assays avoid primer labeling, cumbersome workups, and sophisticated instruments, leading to simpler procedures and improved sensitivity. We expect that these features could lead to major advances in human disease diagnostics and genomic study in the near future.

Detection and differential diagnosis of colon cancer by a cumulative analysis of promoter methylation

“…Compared with conventional methods, the unique features of our method are its high efficiency, simple procedure and low cost 15 . In addition, this method provides a semi-quantitative analysis of differential DNA methylation levels.…”

“…CCPs are highly fluorescent in aqueous media and can amplify the fluorescence intensity of a tightly bound acceptor fluorophore by approximately one order of magnitude through an effective energy transfer pathway 14 . By virtue of the electrostatic interaction between CCP and DNA, extensive optical amplification examples have been demonstrated 15 . Recently, our group has established a simple and highly efficient CCP-based fluorescence resonance energy transfer (FRET) method for analysing gene promoter methylation levels of cancer cells 16 .…”

“…For example, it does not require primer labelling, isolation and washing steps, and is also independent of complicated instruments. Most importantly, our method is suitable for application at the population level because of its sensitivity, simple procedure and label-free characteristics 15,16 . In this paper we established the cumulative methylation alteration panels of multiplex genes using the CCP-based methods, and stepwise discriminant analysis on the basis of the methylation levels of a set of candidate genes in tumour tissues and non-cancerous samples.…”

Synthesis and Characterization of a Water-Soluble Ionic Polyacetylene Derivative

“…Thereby, these materials are important as the active materials in a new generation of electronic and optical devices, including light-emitting devices [15,[19][20][21], photovoltaic cells [22,23], biological and chemical sensors [24,25], photo-detectors [26], optical lasers [27], field-effect transistors [28], and nonvolatile memory devices [29,30].…”

Conjugated Polyelectrolyte (CPE) Poly{3-[6-(N-methylimidazolium)hexyl]-2,5-thiophene} Complexed with DNA: Relation between Colloidal Level Solution Structure and Chromic Effects

“…This allows their use beyond the supramolecular level, for example in nanostructured silica hybrid materials 13 and in thin films prepared through layer-by-layer techniques. 14 Combination of (1) charge transfer as a molecular probe; and (2) water as a non-noxious environment for biomolecules, makes CPEs attractive not only for chemosensors but also for biosensors [15][16][17][18] and protein inactivation. 19 Much of CPE research in biosensors has involved the cationic tetraalkyl-ammonium group which is particularly successful in interrogating negatively charged DNA phosphate linkages.…”