Minggang Deng

Highly Effective Colorimetric and Visual Detection of Nucleic Acids Using an Asymmetrically Split Peroxidase DNAzyme

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.

Conformational Switching of G‐Quadruplex DNA by Photoregulation

Native, self-assembling nucleic acid nanomachines that can walk, move, or rotate have been developed. Owing to their ability to form diverse secondary structures, for example, by the highly sequence-specific hybridization of complementary sequences, the hybridization of DNA and RNA through Watson–Crick H bonds, and the assembly of triplexes through Hoogsteen bonds, nucleic acids are ideal building blocks for the construction of nanodevices. Quadruplex architecture is a nucleic acid secondary structure that plays an important role in nanomachine research, particularly in the control of reversible folding and extension of the G quadruplex of DNA in the presence of external stimuli. Mergny and coworkers reported that a copper(II)-mediated structural switch with a flexible ligand could regulate the conformation of the G quadruplex. Nanodevices based on a quadruplex-toduplex-transition that rely on the use of single-stranded DNA as fuels have been shown to perform rotary movements. Among external stimuli, such as temperature, pH value, electrical-field strength, and molecular recognition, photoregulation is particularly advantageous for controlling movement and conformation. For example, photoregulation does not require any additional components and does not cause undesirable side reactions. Irradiation is an accurate and simple method, and the timing, location, and strength of light can be controlled readily. Moreover, photoregulation provides a clean source of energy and can be repeated many times without loss of efficiency. The introduction of a photochromic group into biomolecules, such as peptides, oligonucleotides, sugar scaffolds, and phospholipids, can cause conformational changes that alter the photochemical properties of the biomolecule. Accordingly, various biological processes involving modified biomolecules can be regulated in a straightforward manner by irradiation. Recently, Ogasawara and Maeda demonstrated the successful photoregulation of G-quadruplex formation through isomerization of a photochromic nucleobase, G, incorporated in aptamers. Spada and co-workers introduced a photoactive moiety at the C8 position of a lipophilic guanosine derivative to regulate the existence of G quartets. However, all these photocontrollers are photochromic modified nucleobases. Specific molecules have not been shown to function as G-quadruplex photocontrollers; thus, we became interested in designing a photoswitch to regulate the formation of G-quadruplex DNA. The azobenzene moiety is widely used as a photoresponsive molecular tool because it possesses excellent photochemical characteristics. Specifically, azobenzene isomerizes to predominantly trans and cis forms under visible (Vis) and ultraviolet (UV) light, respectively. In this study, we synthesized the azobenzene derivative 1 (Scheme 1) to control the movement and conformation of a G quadruplex by irradiation. Our results suggest that the formation and dissociation of G-quadruplex DNAwas induced by interconversion of the trans and cis forms of compound 1. Compound 1 was synthesized by treating 4,4’-dihydroxyazobenzene with 1-(2-chloroethyl)piperidine hydrochloride

The Sensitive and Selective Optical Detection of Mercury(II) Ions by Using a Phosphorothioate DNAzyme Strategy

Guanine-rich DNA sequences are widely found in telomere regions and are prone to forming G-quadruplex structures through cyclic Hoogsteen base pairing between four guanine bases. According to the orientation of oligonucleotides, three formations of G-quadruplex have been suggested: all-parallel, antiparallel, and a hybrid of parallel/antiparallel, all of which have been reported as existing under different conditions. Conformations of the DNA quadruplex were deemed to be affected by several factors such as the composition of the loops, the number of stacked G-tetrads, and the presence of diverse cations or small molecules. Directly modified oligonucleotides, which could be utilized to form a G-quadruplex, have been found, for example, by Hartig and co-workers. The main aim of the study reported herein was to develop a new approach to form G-quadruplex complexes more easily and conveniently by modification of the sequence and to subsequently make use of these conformations as functional probes. Phosphorothioate oligonucleotides are a variant of normal DNA and are characterized by the replacement of a nonbridging oxygen group with a sulfur. This modification facilitates oligonucleotide stability in the presence of endoand exo-nucleases, which could be used in gene regulation through the antisense approach. In addition, recent investigations by Deng and co-workers have indicated that the phosphorothioation of DNA exist in some bacteria, which could be considered as the first known physiological modification on the DNA backbone. To develop this strategy, we combined the two principles that oligonucleotides could be modified with sulfur, and that the formation of phosphorothioate G-quadruplex could be regulated by metal ions, such as Hg , since Hg S forms a strongly covalent bond that would result in the folding of the G-quadruplex. Furthermore, the modified G-quadruplex could be utilized as a functional tool to identify potential targets of anticancer therapy, as well as the detection of specific metal ions or the identification of single-nucleotide polymorphisms (SNPs) as a biological probe. The toxicity of Hg to humans and the environment is associated with its disruption of cell membranes, its impairment of mitochondrial function, and its inhibition of DNA replication in a cell. Up to now, several methods have been developed for the detection of Hg under aqueous conditions with high sensitivity and selectivity, such as the application of DNAzymes and oligonucleotide–gold nanoparticles. The peroxidase-like DNAzyme comprising a G-quadruplex structure and hemin was first designed and developed by Sen and co-workers. Then Willner and co-workers applied it to detect small molecules, metal ions, proteins, and DNA as a catalytic label. Thereafter, Mikuma et al. reported that the tetramolecular G-quadruplex formed by dACHTUNGTRENNUNG(TTAGGG) possessed the ability to bind with hemin. In our strategy, the phosphorothioate modification was adopted within one of the T4G4 sequences, which were inclined to form G-quadruplex structures, and then utilized it to form a DNAzyme (Scheme 1). Compared to the former detection methods of Hg , the utilization of the DNAzyme operated by the phosphorothioate all-parallel G-quadruplex formed from T4G4-S3 combined the advantage of sensitive and selective optical detec[a] D. Zhang, M. Deng, L. Xu, Y. Zhou, J. Yuwen, Prof. X. Zhou College of Chemistry and Molecular Sciences Wuhan University, Hubei Wuhan 430072 (China) Fax: (+86) -27-87336380 E-mail : xzhou@whu.edu.cn [b] Prof. X. Zhou Key Laboratory of Biomedical Polymers of Ministry of Education College of Chemistry and Molecular Sciences Wuhan University, Hubei Wuhan, 430072 (China) [c] Prof. X. Zhou The State Key Laboratory of Natural and Biomimetic Drugs Beijing University (China) [] The two authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901268.

Pyridyl-substituted corrole isomers: synthesis and their regulation to G-quadruplex structures.

G-quadruplex DNA plays an important role in the potential therapeutic target for the design and development of anticancer drugs. As various G-quadruplex sequences in the promoter regions or telomeres can form different secondary structural modes and display a diversity of biology functions, variant G-quadruplex interactive agents may be necessary to cure different disease by differentiating variant types of G-quadruplexes. We synthesize five cationic methylpyridylium corroles and compare the interactions of corroles with different types of G-quadruplexes such as cmyc, htelo, and bcl2 by using surface plasmon resonance. Because of the importance of human telomere G-quadruplex DNA, we focus on the biological properties of the interactions between human telomere G-quadruplex DNA and corrole isomers using CD, T(m), PCR-stop (PCR= polymerase chain reaction), and polymerase-stop assay, which demonstrate the excellent ability of the corrole to induce and stabilize the G-quadruplex. This study provides the first experimental insight into how selectivity might be achieved for different G-quadruplexes by a single group of methylpyridylium corrole isomers that may be optimized for potential selective cancer therapy.

Visual Detection of rpoB Mutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

ABSTRACT Multidrug-resistant Mycobacterium tuberculosis is resistant to two first-line antituberculosis drugs, isoniazid and rifampin, resulting in the relapse of tuberculosis. M. tuberculosis grows very slowly, and thus traditional examination methods take time to test its drug resistance and cannot meet clinical needs. The use of a DNA probe makes it possible to test rifampin resistance. We developed an asymmetrical split-assembly DNA peroxidase assay to detect drug-resistant mutation of rifampin-resistant M. tuberculosis in the rpoB gene rapidly and visibly. A new strategy was also designed to eliminate the adverse effects caused by the complicated secondary structure of the target DNA and to improve the efficiency of the probes. This detection system consists of five group detections, covers rifampin-resistant determination region of the rpoB gene, and tests 40 kinds of mutations, including the most common mutations at codons 531 and 526. Every group detection or individual mutant allele detection can distinguish corresponding mutant DNA sequences from the wild-type DNA sequences.

Studies of the Activity of Peroxidase‐Like DNAzyme by Modifying 3′‐ or 5′‐End of Aptamers

In the presence of hemin and under appropriate conditions, some modalities of G‐quadruplexes can form a peroxidase‐like DNAzyme that has been widely used in biology. Structurefunction studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G‐quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G‐quadruplex sequences by adding different lengths of oligo‐dT to the 3′‐ or 5′‐end of the aptamers. The results suggested that adding dTn to the 5′‐end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dTn to the 3′‐end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H2O2), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.

Site‐Specific Recognition of Guanosine by Manganese(III) Corroles in DNA Non‐Duplex Regions through Active Oxygen Transfer

DNA damage plays an important role in cellular processes. Besides natural protein nucleases, different types of efficient agents for DNA damage have been developed over recent decades in the search for new anticancer and antiviral drugs. In addition to the double-stranded configuration, DNA structures also include some non-duplex regions, which are considered to be from spontaneous errors in DNA replication, thus playing an important role for cells. Herein, we focused on these non-duplex regions of DNA and generated manganese(III) corroles, which exhibit a highly selective cleavage ability for guanosine units located at non-duplex portions, such as loops and bulges. The cleavage mechanism was demonstrated to be a manganese-induced oxidation process. The results given herein show a molecular approach that could specifically probe the guanosine units in DNA non-duplex structures, thus representing a promising step in the construction of tools to target non-duplex structures in chromosomes.

Sensitive and visual detection of adenosine by a rationally designed FokI-based biosensing strategy.

Aptamers are single-stranded nucleic acid molecules that can be screened and isolated using a process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Aptamers can specifically recognize certain targets, such as drugs, ions, amino acids, proteins, and even cells. This unique molecular recognition property has been used to design a number of sensing molecules that can be switched between several distinct conformations. DNA biosensors that offer significant amplification for the detection of DNA have also been developed. Meanwhile, a DNAzyme with a guanine-quartet (G-quartet) structure that binds hemin has demonstrated peroxidase-like activities, and was used to catalyze the oxidation of ABTS (2,2’azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) by H2O2 to produce the colored product ABTS . Owing to the color change observed during the reaction, the DNAzyme can also be used in the design of molecular machine applications. Recently, many groups have developed aptamers as tools for widespread applications in DNA machines and molecular beacons, 14] biotechnology, medical research, amplified sensors, and diagnostics. Adenosine is a product of ATP degradation that is generated during cellular metabolism. Monitoring of adenosine under physiological conditions could be used for practical purposes to further characterize the role of adenosine in both the peripheral and central nervous systems. In 1995, Szostak and co-workers isolated and characterized a DNA aptamer that binds adenosine in solution. Methods for the detection of adenosine based on this aptamer include electrochemical biosensing, affinity chromatography, surface-enhanced Raman scattering, and surveys using quantum dots or fluorescein conjugated to the aptamer. In the present study, we used the aptamer as an input device and DNAzyme as the readout; both of which take the place of detection instruments or probes (in the form of quantum dots or fluorescein). Furthermore, we separated the sequences of the aptamer and DNAzyme into two strands that differ from previous testing models containing the aptamer and DNAzyme in the same strand. FokI acts as the modulation molecule to monitor the signal transfer between the strands. FokI is an unusual restriction enzyme in that it recognizes the cognate sequence 5’-GGATG-3’ and cleaves the DNA phosphodiester group 9 bp from this sequence and 13 bp away from the complementary strand. In previous studies, the restriction enzyme FokI along with the DNA digestion idea has been employed for an analyte gene sequence, but not for small molecules. 20] Currently, FokI plays a more important role in managing the message transporter as a result of the different positions of the recognition site and cleavage site. Here, we design a FokI-based DNA biosensor that employs the aptamer (OD1) as the input detection device, one oligonucleotide (OD2) as a message transporter, DNAzyme (OD4) as the resultant output device, the remaining oligonucleotide (OD3) as the repressor of DNAzyme, and FokI as the manager of the different functional oligomers. The transporter (OD2) contains the sequence 5’-GGATG-3’, and the repressor (OD3) possesses another cleavage site: 5’CATCC-3’. Both sites play an important role in releasing the DNAzyme (OD4). Scheme 1 illustrates the operation principle of the FokI-based aptamer-DNAzyme biosensor, which includes three steps: A) adenosine, as an input signal, binds to the aptamer (OD1) then allows the transporter [a] M. Zhang, J. Huang, Dr. M. Deng, Dr. X. Weng, H. Ma, Prof. X. Zhou College of Chemistry and Molecular Sciences Ministry of Education, Key Lab Biomed State Key Laboratory of Virology Wuhan University Wuhan, Hubei, 430072 (P.R. China) Fax: (+86) 27-8733-6380 E-mail : xzhou@whu.edu.cn [b] Prof. X. Zhou State Key Laboratory of Natural and Biomimetic Drugs Beijing University State Key Laboratory of Applied Organic Chemistry Lanzhou University Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.200900200.

Quadruplex-Duplex Motifs as New Topoisomerase I Inhibitors

In this article, 13 short chains that can form G-quadruplex and quadruplex-duplex motif have been designed. Fourteen oligonucleotides, including 13 short chains as well as a reference short chain all have certain level of inhibition to topoisomerase I, whether or not they form G-quadruplex and quadruplex-duplex motif, and the G-quadruplex and quadruplex-duplex motif show better activity than single short chain. The result confirmed that after forming G-quadruplex and quadruplex-duplex motif these 14 oligonucleotides are competitive inhibition, that is, through the priority binding with the topoisomerase I and precluding from its binding with the normal substrate pBR322 and, therefore, inhibiting the next reaction.

A pyridyl carboxamide molecule selectively stabilizes DNA G-quadruplex and regulates duplex–quadruplex competition

G-Quadruplexes formed by G-rich DNA are of broad interest due to their involvement in telomere function, gene transcription and recombination. Small ligands that interact strongly with G-quadruplexes have been considered to further influence telomeric function and gene transcription. Because most G-rich sequences are trapped in duplex structures in gene promoters, ligands that can stabilize G-quadruplexes in the presence of their complimentary strands would likely have strong effects on gene transcription. Here, we report a novel simple small molecule (pyridyl carboxamide), consisting of three pyridine rings and four amide bonds. Comparing with some reported G-quadruplex ligands, this molecule not only selectively stabilizes G-quadruplexes rather than duplexes, but also maintains a G-quadruplex structure even if the G-rich region was trapped in long double-stranded DNA (dsDNA). It is widely believed that the dissociation of duplexes is involved in gene transcription and that the formation of the G-quadruplex influences some oncogene expression. Py-Am exhibited strong G-quadruplex-forming ability within a long dsDNA sequence, suggesting it would have potent effects on the G-quadruplex-forming sequences involved in gene transcription.