Shengyong Yan

Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy

Existence of G-quadruplex DNA in vivo always attract widespread interest in the field of biology and biological chemistry. We reported our findings for the existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Probes for selective G-quadruplex cross-linking was designed and synthesized that show high selectivity for G-quadruplex cross-linking. Further biological studies demonstrated its good inhibition activity against murine melanoma cells. To further investigate if G-quadruplex DNA was formed in vivo and as the target, a derivative was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll 70 and PEG, was used to investigate the compounds pure cross-linking ability upon preformed G-quadruplex. Thus, as a potent G-quadruplex cross-linking agent, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy.

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

Selective chemical labelling of 5-formylcytosine in DNA by fluorescent dyes.

Direct labelling: 5-Formylcytosine in DNA can be selectively labelled by fluorescent dyes containing an active amino group. The labelled DNA shows strong fluorescence and can be detected by polyacrylamide gel electrophoresis (PAGE) and fluorescence measurements (see scheme). This method can distinguish 5-formylcytosine from other methylation forms of cytosine in DNA.

Selective detection of 5-formyl-2'-deoxycytidine in DNA using a fluorogenic hydroxylamine reagent.

Fluorogenic hydroxylamine reagents were used for detecting 5-fC through a labeling pathway. Chemical synthesis, HPLC, denaturing PAGE, and DNA MS were applied to testify that the probe reacted with 5-fC with oligodeoxynucleotide selectivity to achieve 5-fC detection conveniently and quantificationally with the method of fluorescence. The feasibility of fluorescently detecting 5-fC in a genome was also investigated.

A two-photon fluorescent probe for intracellular detection of tyrosinase activity.

AAN effective sensor: The two-photon turn-on fluorescent probe NHU was synthesized to optically detect tyrosinase activity in vitro and in melanoma cells. NHU is composed of a 4-aminophenol moiety and a naphthylamine unit, both of which are connected through a urea linkage. Upon exposure to tyrosinase, the 4-aminophenol site is gradually oxidized to the corresponding orthoquinone, ultimately releasing the highly fluorescent product 6-acyl-N-methyl-2-naphthylamine (AAN).

A turn-on fluorescent probe for detection of tyrosinase activity

We have presented a fluorescent probe that exhibits a fluorescence turn-on signal upon reaction with tyrosinase, and we show that it can be readily employed for the assessment of tyrosinase activity and tyrosinase inhibitor activities in buffered aqueous solution.

Nonlinear optical dye TSQ1 as an efficiently selective fluorescent probe for G-quadruplex DNA

A squarylium dye TSQ1 shows a remarkable fluorescence enhancement selectivity for the G-quadruplex DNA structure against duplex DNA, it can be distinguished even by the naked eye. What is more important, TSQ1 did not induce the G-rich sequence folding into G-quadruplex structure which means TSQ1 can be used to detect G-quadruplex structures in cells.

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.

A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging.

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258, our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT assays.

Graphene oxide-based fluorescent detection of DNA and enzymes using Hoechst 33258 and its use for dual-output fluorescent logic gates

Based on graphene oxide and Hoechst 33258, bioassays towards target DNA and several enzymes including Exo III and Dam MTase were established. This strategy eliminates the need for modification of oligonucleotides, and can give similarly satisfactory results compared to traditional methods using labelled DNA. Moreover, “AND” and “INHIBIT” dual-output DNA logic gates were developed.