Bifunctional Colorimetric Oligonucleotide Probe Based on a G-Quadruplex DNAzyme Molecular Beacon

A label-free bifunctional colorimetric oligonucleotide probe for DNA and protein detection has been developed on the basis of a novel catalytic molecular beacon consisting of two hairpin structures and a split G-quadruplex DNAzyme in the middle. The two loops of this molecular beacon consist of thrombin aptamer sequence and the complementary sequence of target DNA, which are utilized to sense single-stranded DNA and thrombin. The G-quadruplex DNAzyme can effectively catalyze the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine sulfate to generate colorimetric signal. Upon addition of the target, the DNA or protein combines with one loop of the hairpin structures, and meanwhile drives the middle G-quadruplex DNAzyme to dissociate. This results in a decrease of catalytic activity, enabling the separate analysis of DNA and thrombin

Four-Way Junction-Driven DNA Strand Displacement and Its Application in Building Majority Logic Circuit

We introduced a four-way DNA junction-driven toehold-mediated strand displacement method. Separation of the different functional domains on different strands in the four-way junction structure and usage of glue strand to recombine them for different logic gates make the design more flexible. On the basis of this mechanism, a majority logic circuit fabricated by DNA strands was designed and constructed by assembling three AND gates and one OR gate together. The output strand drew the G-rich segments together to form a split G-quadruplex, which could specifically bind PPIX and enhance its fluorescence. Just like a poll with three voters, the high fluorescence signal would be given off only when two or three voters vote in favor. Upon slight modification, the majority circuit was utilized to select the composite number from 0 to 9 represented by excess-three code. It is a successful attempt to integrate the logic gates into a circuit and to achieve desired functions

Simple and Sensitive Fluorescent and Electrochemical Trinitrotoluene Sensors Based on Aqueous Carbon Dots

Aqueous N-rich carbon dots (CDs), prepared by the microwave-assisted pyrolysis method, are applied as a dual sensing platform for both the fluorescent and electrochemical detection of 2,4,6-trinitrotoluene (TNT). The fluorescent sensing platform is established on the strong TNT–amino interaction which can quench the photoluminescence of amino functionalized CDs through charge transfer. The resultant linear detection ranges from 10 nM to 1.5 μM with a fast response time of 30 s. Glassy carbon electrode modified with CDs exhibits a fine capability for TNT reduction with the linear range from 5 nM to 30 μM, better than that obtained by the fluorescent method. Moreover, the minimum distinguishable response concentration with respect to these two methods is down to the nanomolar level with a high specificity and sensitivity

Aptamer-Based Sensing Platform Using Three-Way DNA Junction-Driven Strand Displacement and Its Application in DNA Logic Circuit

We proposed a new three-way DNA junction-driven strand displacement mode and fabricated an aptamer-based label-free fluorescent sensing platform on the basis of this mechanism. Assembling the aptamer sequence into the three-way DNA junction makes the platform sensitive to the target of the aptamer. A label-free signal readout method, split G-quadruplex enhanced fluorescence of protoporphyrin IX (PPIX), was used to report the final signal. Here, adenosine triphosphatase (ATP) was taken as a model and detected through this approach, and DNA strand could also be detected by it. The mechanism was investigated by native polyacrylamide gel electrophoresis. Furthermore, on the basis of this molecular platform, we built a logic circuit with ATP and DNA strands as input. Aptamer played an important role in mediating the small molecule ATP to tune the DNA logic gate. Through altering the aptamer sequence, this molecular platform will be sensitive to various stimuli and applied in a wide field

G‑quadruplex-Based Fluorescent Assay of S1 Nuclease Activity and K⁺

Endonuclease plays an important role in many biological processes, and an assay of endonuclease activity is of great significance. However, traditional methods for the assay of endonuclease activity have undesirable limitations, such as high cost, DNA-consuming and laboriousness. In the present work, a G-quadruplex-based, fluorescent assay of endonuclease activity has been developed with protoporphyrin IX (PPIX) as a signal reporter. S1 nuclease, a single strand DNA (ssDNA)-specific endonuclease, is employed as model system. In the “on” state, G-quadruplex DNA can greatly enhance the fluorescence of PPIX. However, if S1 nuclease could cleave G-quadruplex DNA into small fragments, there would be no formation of G-quadruplexes, accompanied by low emission response of PPIX. This fluorescent discrimination before or after digestion by nuclease can be used to monitor the activity of S1 nuclease. This assay is simple in design and offers a convenient protocol for homogeneous, rapid and high-throughput detection. In addition, the proposed strategy avoids complicated covalent modifications or chemical labeling, and thus offers advantages of simplicity and cost efficiency. More importantly, K⁺ is found to well inhibit the activity of S1 nuclease when using certain G-quadruplex DNA as substrate, and thus this system is further used for turn-on detection of K⁺. S1 nuclease is critical in the detection of K⁺ since it helps to reduce the background signal

Photoinduced Electron Transfer of DNA/Ag Nanoclusters Modulated by G‑Quadruplex/Hemin Complex for the Construction of Versatile Biosensors

Photoinduced electron transfer (PET) has been observed for the first time between DNA/Ag fluorescent nanoclusters (NCs) and G-quadruplex/hemin complexes, accompanied by a decrease in the fluorescence of the DNA/Ag NCs. In this PET process, a parallel G-quadruplex and the sensing sequences are blocked by a duplex. The specific combination of targets with the sensing sequence triggers the release of the G-quadruplex and allows it to fold properly and bind hemin to form a stable G-quadruplex/hemin complex. The complex proves favorable for PET because it makes the G-quadruplex bind hemin tightly, which promotes the electron transfer from the DNA/Ag NCs to the hemin Fe^III center, thus resulting in a decrease in the fluorescence intensity of the DNA/Ag NCs. This novel PET system enables the specific and versatile detection of target biomolecules such as DNA and ATP with high sensitivity based on the choices of different target sequences

Lighting Up the Thioflavin T by Parallel-Stranded TG(GA)<i>n</i> DNA Homoduplexes

Thioflavin T (ThT) was once regarded to be a specific fluorescent probe for the human telomeric G-quadruplex, but more other kinds of DNA were found that can also bind to ThT in recent years. Herein, we focus on G-rich parallel-stranded DNA and utilize fluorescence, absorbance, circular dichroism, and surface plasmon resonance spectroscopy to investigate its interaction with ThT. Pyrene label and molecular modeling are applied to unveil the binding mechanism. We find a new class of non-G-quadruplex G-rich parallel-stranded (<i>ps</i>) DNA with the sequence of TG­(GA)<i>n</i> can bind to ThT and increase the fluorescence with an enhancement ability superior to G-quadruplex. The optimal binding specificity for ThT is conferred by two parts. The first part is composed of two bases TG at the 5′ end, which is a critical domain and plays an important role in the formation of the binding site for ThT. The second part is the rest alternative d­(GA) bases, which forms the <i>ps</i> homoduplex and cooperates with the TG bases at the 5′ end to bind the ThT

Graphene-Based Aptamer Logic Gates and Their Application to Multiplex Detection

In this work, a GO/aptamer system was constructed to create multiplex logic operations and enable sensing of multiplex targets. 6-Carboxyfluorescein (FAM)-labeled adenosine triphosphate binding aptamer (ABA) and FAM-labeled thrombin binding aptamer (TBA) were first adsorbed onto graphene oxide (GO) to form a GO/aptamer complex, leading to the quenching of the fluorescence of FAM. We demonstrated that the unique GO/aptamer interaction and the specific aptamer–target recognition in the target/GO/aptamer system were programmable and could be utilized to regulate the fluorescence of FAM <i>via</i> OR and INHIBIT logic gates. The fluorescence changed according to different input combinations, and the integration of OR and INHIBIT logic gates provided an interesting approach for logic sensing applications where multiple target molecules were present. High-throughput fluorescence imagings that enabled the simultaneous processing of many samples by using the combinatorial logic gates were realized. The developed logic gates may find applications in further development of DNA circuits and advanced sensors for the identification of multiple targets in complex chemical environments

Engineering DNA Three-Way Junction with Multifunctional Moieties: Sensing Platform for Bioanalysis

Functionalization of DNA nanostructures is critical to the achievement of the application in biosensors. Herein, we demonstrate a novel DNA three-way junction (TWJ) with three functional moieties, which integrates the electrochemical, fluorescent, and colorimetric properties. Upon addition of external stimuli, including DNA, thrombin, and ATP, the specific interactions between targets and sensing elements could induce strand displacement reaction and conformation transformation, resulting in the integration of G-quadruplex/hemin complex as electrochemical probe, lighting up the fluorescence of DNA/Ag nanoclusters and enhancing the catalytic activity of DNAzyme to catalyze the H₂O₂–TMB system to generate colorimetric signal. Such a functional DNA nanostructure actually serves as a biosensing platform, showing high sensitivity and selectivity for DNA, thrombin, and ATP detection. Furthermore, We also show that this novel sensing platform can be utilized to detect three different kinds of targets independently and simultaneously. Our integrated concept provides a paradigm for exploring the potential of TWJs in analytical applications