G-quadruplex-based DNAzyme for facile colorimetric detection of thrombin

Thrombin-binding aptamer is found to bind hemin to form a catalytic complex whose activity is significantly promoted by the addition of thrombin, which enables the colorimetric detection of thrombin with high specificity and sensitivity in a facile way.

Chemiluminescence thrombin aptasensor using high-activity DNAzyme as catalytic label

With a designed high-activity DNAzyme as the catalytic label, an ultrasensitive chemiluminescence thrombin aptasensor is developed, enabling a 10- to 100-fold improvement in the detection sensitivity as compared with previous counterparts.

DNAzyme-based Colorimetric Sensing of Lead (Pb2+) Using Unmodified Gold Nanoparticle Probes

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, have been intensively studied due to their potential applications in therapeutics and sensors. Taking advantage of the high specificity of 17E DNAzyme for Pb2+, highly sensitive and selective fluorescent, electrochemical and colorimetric sensors have been developed for Pb2+. In this work, we report a simple, sensitive and label-free 17E DNAzyme-based sensor for Pb2+ detection using unmodified gold nanoparticles (GNPs) based on the fact that unfolded single-stranded DNA could be adsorbed on the citrate protected GNPs while double-stranded DNA could not. By our method the substrate cleavage by the 17E DNAzyme in the presence of Pb2+ could be monitored by color change of GNPs, thereby Pb2+ detection was realized.

G-quadruplex-based DNAzyme for sensitive mercury detection with the naked eye

Hg2+ is able to inhibit the peroxidase-like DNAzyme function of a T-containing G-quadruplex DNA via Hg2+-mediated T-T base pairs, which enables the visual detection of Hg2+ in the TMB-H2O2 reaction system with high selectivity and sensitivity.

Investigation of 3,3 ',5,5 '-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme

in this Work, the suitability of 3,3',5,5'-tetramethylbenzidine sulfate (TMB) as the substrate of a DNAzyme catalytic system composed of a guanine-quadruplex DNA molecule and hemin was investigated. In the presence of H2O2, the hemin-DNA complex catalyzes the oxidation of TMB to produce two colored products, much like a peroxidase. The color-generating activity of this system could be influenced by several factors such as buffer type, pH value, DNA sequence, reaction time, and concentrations of both the hemin and H2O2. To illustrate the utility of this catalytic system, we designed a colorimetric assay, in which a synthetic oligonucleotide with a sequence complementary to the G-quadruplex DNA was used as the target. A detection limit of 1.86 nM was obtained. Our data have shown that TMB was an excellent colorimetric indicator that reported the peoxidase activities of the widely studied hemin-G-quadruplex DNAzyme system.

G-Quadruplex Aptamers with Peroxidase-Like DNAzyme Functions: Which Is the Best and How Does it Work?

Some G-quadruplex DNA aptamers have been found to strongly bind hemin to form DNAzymes with peroxidase-like activity. To help determine the most suitable DNAzymes and to understand how they work, five previously reported G-quadruplex aptamers were compared for their binding affinity and then the potential catalytic mechanism of their corresponding hemin-G-quadruplex DNAzymes was explored. Among these aptamers, a G-quadruplex named AGRO100 was shown to possess the highest hemin-binding affinity and the best DNAzyme function. This means that AGRO100 is the most ideal candidate for DNAzyme-based analysis. Furthermore, we found the peroxidase-like activity of DNAzyme to be primarily dependent on the concentration of H2O2 and independent of that of the peroxidase substrate (that is, 2,2-azino-bis(3-ethytbenzothiazoline-6-sulfonic acid)diammonium salt). Accordingly, a reaction mechanism for DNAzyme-catalyzed peroxidation is proposed. This study provides new insights into the G-quadruplex-based DNAzymes and will help us to further extend their applications in the analytical field.

Enhanced catalytic DNAzyme for label-free colorimetric detection of DNA

A DNAzyme-based label-free method for the colorimetric detection of DNA is introduced, with a supramolecular hemin G-quartet complex as the sensing element and a 36-mer single-strand DNA as the analyte that is detected at 10 nM.

Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.