Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells.

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 μM TS primer (5'-AATCCGTCGAGCAGAGTT-3') and 0.

A mRNA-Responsive G-Quadruplex-Based Drug Release System.

G-quadruplex-based drug delivery carriers (GDDCs) were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II) phthalocyanine (CuAPC). As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR) mRNA.

A simple "add and measure" FRET-based telomeric tandem repeat sequence detection and telomerase assay method.

A simple and sensitive method for measuring telomeric tandem repeat DNA and telomerase activity based on fluorescence resonance energy transfer (FRET) with a FAM-modified 12-mer ODN probe as a donor (fluorophore) and ethidium bromide (EB) as an acceptor (quencher) is proposed. When telomeric DNA and the FAM-modified probe form a duplex, EB intercalates between base-pairs, resulting in fluorescence quenching of FAM through FRET from FAM to EB. This method can be used to estimate the amount of telomeric DNAs in a sample solution as the molar concentration of the telomeric DNA unit [5'-(TTA GGG TTA GGG)-3'].

In vitro assays predictive of telomerase inhibitory effect of G-quadruplex ligands in cell nuclei.

G-quadruplex-binding and telomerase-inhibiting capacities of G-quadruplex ligands were examined under a cell nuclei-mimicking condition including excess double-stranded DNA (λ DNA) and molecular crowding cosolute (PEG 200). Under the cell nuclei-mimicking condition, a cationic porphyrin (TMPyP4) did not bind to the G-quadruplex despite the high affinity (Ka = 3.6 × 10(6) M(-1)) under a diluted condition without λ DNA and PEG 200.

A highly sensitive telomerase activity assay that eliminates false-negative results caused by PCR inhibitors.

An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA.

Multiple and cooperative binding of fluorescence light-up probe thioflavin T with human telomere DNA G-quadruplex.

Thioflavin T (ThT), a typical probe for protein fibrils, also binds human telomeric G-quadruplexes with a fluorescent light-up signal change and high specificity against DNA duplexes. Cell penetration and low cytotoxicity of fibril probes having been widely established, modifying ThT and other fibril probes is an attractive means of generating new G-quadruplex ligands. Thus, elucidating the binding mechanism is important for the design of new drugs and fluorescent probes based on ThT.

Study on effects of molecular crowding on G-quadruplex-ligand binding and ligand-mediated telomerase inhibition.

The telomere G-quadruplex-binding and telomerase-inhibiting capacity of two cationic (TMPyP4 and PIPER) and two anionic (phthalocyanine and Hemin) G-quadruplex-ligands were examined under conditions of molecular crowding (MC). Osmotic experiments showed that binding of the anionic ligands, which bind to G-quadruplex DNA via π-π stacking interactions, caused some water molecules to be released from the G-quadruplex/ligand complex; in contrast, a substantial number of water molecules were taken up upon electrostatic binding of the cationic ligands to G-quadruplex DNA. These behaviors of water molecules maintained or reduced the binding affinity of the anionic and the cationic ligands, respectively, under MC conditions.

Specific binding of anionic porphyrin and phthalocyanine to the G-quadruplex with a variety of in vitro and in vivo applications.

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin).

Phthalocyanines: a new class of G-quadruplex-ligands with many potential applications.

A G-quadruplex is a four-stranded DNA structure featuring stacked guanine tetrads, G-quartets. Formation of a G-quadruplex in telomere DNA can inhibit telomerase activity; therefore, development of G-quadruplex-ligands, which induce and/or stabilize G-quadruplexes, has become an area of great interest. Phthalocyanine derivatives have substantial potential as high-affinity G-quadruplex-ligands because these planar chromophores are similar in size and shape to the G-quartets.

Anionic phthalocyanines targeting G-quadruplexes and inhibiting telomerase activity in the presence of excessive DNA duplexes.

Anionic phthalocyanines inhibited efficiently telomerase activity even in the presence of excess double-stranded DNA (dsDNA) because of their selective binding to telomere G-quadruplex, although low selectivity of a typical cationic ligand, TMPyP4, to the G-quadruplex allowed telomerase reaction under conditions with dsDNA.

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem.

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases.