Site-Specific Discrimination of Cytosine and 5-Methylcytosine in Duplex DNA by Peptide Nucleic Acids

For site-specific discrimination of cytosine (C) and 5-methylcytosine (mC) in duplex DNA, we developed a new method using peptide nucleic acids (PNAs). The combination of a PNA-assisted DNA displacement complex and a fluorescein-labeled probe oligomer allowed the detection of mC at the defined sites in target DNA using a restriction enzyme. After treatment of the complex with a restriction enzyme, strong fluorescence emission was observed for the complex containing C at the target site, whereas the fluorescence intensity for the complex containing mC was extremely weak

Synthesis and Properties of Peptide Nucleic Acids Containing a Psoralen Unit

We prepared the psoralen PNA unit from 8-methoxypsoralen and synthesized various PNAs containing psoralen by a typical tBoc method. PNAs containing psoralen (P-PNA) at strand end formed a stable duplex with complementary DNA. The hybridization of P-PNA with complementary DNA resulted in a considerable decrease of the psoralen fluorescence

Surface-Enhanced Raman Scattering Identification of Nucleic Acid Targets by Acetylene-Tagged Hoechst Molecule Binding with DNA-Tethered Gold Nanoparticles

Raman probes have been utilized to identify biomolecules. However, their application for nucleic acid sensing remains limited because of the requirement of purification processes. Here, we provide a general purification-free method for the detection of specific nucleic acids based on surface-enhanced Raman scattering (SERS) using functionalized gold nanoparticles. We identified the robust Raman signal of an acetylene-tagged Hoechst molecule (AH) through SERS because of its binding to nanoparticles bearing double-stranded oligodeoxynucleotides on their surface. Once target nucleic acids were added to the system, strand displacement on the particles and dissociation of AH from the particles occurred spontaneously, leading to a dramatic decrease in the signal intensity. We also found that the cellular miRNA level was successfully identified by the system

One-Electron Photooxidation and Site-Selective Strand Cleavage at 5-Methylcytosine in DNA by Sensitization with 2-Methyl-1,4-naphthoquinone-Tethered Oligonucleotides

Photosensitized one-electron oxidation was applied to discriminate a specific base site of 5-methylcytosine (mC) generated in DNA possessing a partial sequence of naturally occurring p53 gene, using a sensitizing 2-methyl-1,4-naphthoquinone (NQ) chromophore tethered to an interior of oligodeoxynucleotide (ODN) strands. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of mC-containing DNA and NQ-tethered complementary ODN led to oxidative strand cleavage selectively at the mC site, when the NQ chromophore was arranged so as to be in close contact with the target mC. The target mC is most likely to be one-electron oxidized into the radical cation intermediate by the sensitization of NQ. The resulting mC radical cation may undergo rapid deprotonation and subsequent addition of molecular oxygen, thereby leading to its degradation followed by strand cleavage at the target mC site. In contrast to mC-containing ODN, ODN analogs with replacement of normal cytosine, thymine, adenine, or guanine at the mC site underwent less amount of such an oxidative strand cleavage at the target base site, presumably due to occurrence of charge transfer and charge recombination processes between the base radical cation and the NQ radical anion. Furthermore, well designed incorporation of the NQ chromophore into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. Thus, photosensitization by an NQ-tethered ODN led to one-electron oxidative strand cleavage exclusively at the target mC site, providing a convenient method of discriminating mC in naturally occurring DNA such as human p53 gene as a positive band on a sequencing gel

Fluorometric Identification of 5-Methylcytosine Modification in DNA: Combination of Photosensitized Oxidation and Invasive Cleavage

An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine (mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission

One-Electron Reductive Template-Directed Ligation of Oligodeoxynucleotides Possessing a Disulfide Bond

One-Electron Reductive Template-Directed Ligation of Oligodeoxynucleotides Possessing a Disulfide Bon

Raman Signal Enhancement by DABCYL-Substitution on DNA Aptamer for Identification of Cellular ATP

Raman probes have attracted widespread attention for the visualization and identification of biomolecules, because they can be applied to identify detailed chemical structures, detect multiple molecules simultaneously, and visualize cellular functional molecules. However, the biological application of Raman probes is still limited because of their weak signal intensity. Herein, we present a molecular system that shows an enhanced Raman signal using a nonfluorescent dye. We introduced a DABCYL molecule bearing an acetylene unit into thymidine at the 5-position. The resulting modified nucleobase, dDAU, showed a robust signal around 2200 cm–1, which was attributed to the acetylene unit, due to resonance Raman induced by the DABCYL group. We further prepared a DNA aptamer modified with dDAU, and characterized the change of the Raman spectra. Combination with gold nanoparticles, which enhanced the Raman signal by surface-enhanced Raman scattering (SERS), allowed sensitive detection of cellular adenosine derivatives including ATP. Thus, the present system is a promising tool for the detection of biological materials by Raman spectroscopy

Enzyme-Catalyzed Conversion of Chemical Structures on the Surface of Gold Nanorods

For developing metal nanoparticles of which surface chemical structures could be altered by enzymes in the cells, functional linkers caged by coumaric acids have been synthesized. Synthesized gold nanorod (GNR) conjugates possessing coumaric acid precursors underwent (porcine liver) esterase-catalyzed hydrolysis on their surface to afford GNRs coated with amino-functionalized polyethylene glycol and fluorescent coumarins as reporter molecules for monitoring the conversion. The chemical structural conversion on the GNR surfaces was successfully observed inside cells by fluorescence microscopy when GNR conjugates were incubated with tumor cells

Site Selective Formation of Thymine Glycol-Containing Oligodeoxynucleotides by Oxidation with Osmium Tetroxide and Bipyridine-Tethered Oligonucleotide

Site Selective Formation of Thymine Glycol-Containing Oligodeoxynucleotides by Oxidation with Osmium Tetroxide and Bipyridine-Tethered Oligonucleotid