Cationic Conjugated Polymer/DNA Complexes for Amplified Fluorescence Assays of Nucleases and Methyltransferases

Abstract

In recent years, it has become a major goal to develop new techniques capable of screening protein-based DNA cleavage agents in modern drug discovery and in the molecular biology field. Among DNA cleaving agents, restriction or nonrestriction endonucleases are involved in many important biological processes, such as DNA replication, recombination, and repair, [1][2][3] and the DNA cleavage by them is extensively utilized as a necessary tool in polymerase chain reaction (PCR) assays, gene mapping, molecular cloning, and medicinal chemistry. [4,5] Traditional methods have been established in assaying nuclease activities, such as gel electrophoresis, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). [6][7][8][9] These methods share the drawbacks of being time consuming, DNA consuming, the necessity of substrate radiolabeling, or laborious workup treatment.As a way to circumvent the limitations, highly sensitive and convenient "molecular break light" probes based on a fluorescence-quenching technique have been developed for assaying restriction endonuclease with a double-stranded DNA (dsDNA) substrate. [10] Recently an alternative assay using a "molecular beacon" has also been reported for assaying nonspecific nuclease with a single-stranded DNA (ssDNA) substrate.[11] However, they require doubly labeled DNA probes, which are expensive. Furthermore, the sensing signals used in these studies are based on intensity measurements, which are easily affected by external nonspecific events. In this respect, the fluorescence resonance energy transfer (FRET) technique exhibits a big shift in emission profiles and provides a ratiometric fluorescence measurement to reduce the nonspecific interaction events. [12] To date, only a few studies have demonstrated the assaying of nuclease activity using FRET probes. [13,14] However, they also require expensive doubly labeled DNA probes. Thus it still remains a challenge to find a ratiometric, sensitive and convenient approach to assay restriction and nonrestriction nucleases. Conjugated polymers (CPs) coordinate the action of a large number of absorbing units with efficient intrachain and interchain energy-transfer mechanisms. [15,16] The transfer of excitation energy along the whole backbone of the CP to an energy/ electron acceptor results in an amplification of the fluorescence signals. Therefore, CPs can be used in the function of very sensitive optical detections for bio-macromolecules, such as nucleic acids and proteins. [17][18][19][20][21][22][23][24][25][26][27][28][29] Recently, we reported a colorimetric method using cationic polythiophene derivatives initialized by the Leclerc group [20] to monitor the cleavage of ssDNA by single-strand-specific S1 nuclease. [30] One disadvantage of this approach is that it doesn't work for restriction nucleases with dsDNA substrates because the polymer can not recognize the cleaved small ssDNA fragments in the presence of dsDNA ones; the other is that the UV-based measurement imparts the assay l...

Chemical sensors based on π-conjugated organic molecules and gold nanoparticles

“…An additional advantage of using polymer as sensor materials emerge from the modular nature of polymers; that is, the structure and sequence of the repeating units within polymers can be widely varied and modified, which allows the polymers to be tailored to suit for diverse targets and potentially achieve high selectivity. Therefore, conjugated polymers can be used as an optical platform in highly selective chemical and biological sensors [94][95][96][97] . Recently, a variety of conjugated polymers containing molecular recognition sites have been reported as sensing materials for metal ions [98,99] .…”

Fluorescent Conjugated Polyelectrolytes for Biomacromolecule Detection

“…Reaction B in Figure 2a gives a turn-off response for a FRET event, and is designed to detect DNA cleavage by nucleases. [17] DNA was cleaved by the nuclease near the 5 0 -terminus where fluorescein was labeled. After digestion the fluoresceincontaining fragment binds to P3 very weakly in 200 Â 10 À3 M phosphate buffer saline (PBS, pH 7.4) as compared with uncleaved DNA, which results in inefficient FRET.…”

Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon

“…[16][17][18][19][20][21][22] The use of complexes of DNA and cationic conjugated polymer as probes was recently described. [23] Colorimetric methods based on enzyme-responsive gold nanoparticle (AuNP) [24][25][26][27] and DNAzyme [28] systems have also been developed for detecting endonucleases. Although each of these techniques has its own advantages, most of them do not afford multiplex detection of endonucleases.…”