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Assembly of DNA structures based on hybridization like split G-quadruplex (GQ) have great potential for the base-pair specific identification of nucleic acid targets. Herein, we combine multiple split G-quadruplex (GQ) assemblies on designed DNA nanostructures (carrier) with a solid-state nanopore sensing platform. The split GQ probes recognize various nucleic acid sequences in a parallel assay that is based on glass nanopore analysis of molecular structures. Specifically, we split a GQ into two asymmetric parts extended with sequences complementary to the target. The longer G-segment is in solution, and the shorter one is on a DNA carrier. If the target is present, the two separate GQ parts will be brought together to facilitate the split GQ formation and enhance the nanopore signal. We demonstrated detection of multiple target sequences from different viruses with low crosstalk. Given the programmability of this DNA based nanopore sensing platform, it is promising in biosensing.

G-quadruplexes (Gqs) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ∼5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers, establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un)folded Gq structures, provides insights into single-molecule kinetics of Gq folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of Gqs at the single-molecule level without labeling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.

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 ( ps) DNA with the sequence of TG(GA) n 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 ps homoduplex and cooperates with the TG bases at the 5' end to bind the ThT.

Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.

A novel visible multi-digit DNA keypad lock system was fabricated based on split G-quadruplex DNAzyme and silver microspheres. The final result of the keypad lock can be easily recognized by the naked eye and the number of inputs for the keypad lock can be flexibly adjusted. This molecular platform showed excellent scalability and flexibility.

Adopting fluorescence of PPIX enhanced by a split G-quadruplex and toehold mediated strand displacement reaction, a series of unlabeled fluorescent logic gates is set up and some of them are cascaded into circuits. Controlled release of PPIX, which is also a photosensitizer in photodynamic diagnosis and therapy, is realized by this circuit, making it a wise choice for DNA computing., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

An effective G-quadruplex-based probe has been constructed for rapid and sensitive detection of Cu(2+). In this probe, an anionic porphyrin, protoporphyrin IX (PPIX) served as a reference signal, which binds to G-quadruplex specifically and the fluorescence intensity increases sharply. While, in the presence of Cu(2+), the G-quadruplex can catalyze the related Cu(2+) insertion into the protoporphyrin, the fluorescent intensity is decreased. The fluorescence of the response ligand could be selectively quenched in the presence of Cu(2+) and not interfered by other metal ions. The probe provided an effective platform for reliable detection of Cu(2+) with a detection limit as low as 3.0nM, the high sensitivity was attributed to the strong metalation of PPIX with Cu(2+) catalyzed by G-quadruplex (PS5.M). Linear correlations were obtained over the logarithm of copper ion concentration in the range from 8×10(-9)M to 2×10(-6)M (R=0.998). The G-quadruplex-based probe also could be used to detect Cu(2+) in real water samples. Additionally, these striking properties endow the G-quadruplex-ligand with a great promise for analytical applications., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A special DNA calliper made of a split G-quadruplex was constructed to measure the length of DNA in a single-digit base number range between two selected sequences. The enhanced fluorescence of PPIX changed with the distance between two G-rich segments.

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 H(2)O(2)-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.

Efficient and economic DNA nanomaterials that can work as logic components are necessary for the development of DNA computers with high speed and outstanding data storage capacity. A new molecular catalytic beacon (MCB) and a series of label-free colorimetric logic gates based on the formation and dissociation of G-quadruplex DNAzyme were established in this work. These logic gates (NOT, NOR, IMPLICATION, AND, OR and INHIBIT) were realized by the interaction between the special designed oligonucleotide hairpins and the short input single strand complementary DNA. We were able to recognize the logic output signals effortlessly by our naked eyes. It is a simple, economic and safe approach for the design of complex multiple input DNA logic molecular device., (Copyright © 2011 Elsevier Ltd. All rights reserved.)