G-rich DNA Research Articles

Synthesis, G-Quadruplex DNA binding and cytotoxic properties of naphthalimide substituted styryl dyes

G-Quadruplex DNAs, formed by G-rich DNA sequences in human genes, are promising targets for design of cancer drugs. In this study, two naphthalimide substituted styryl dyes with different sizes of aromatic groups were synthesized. The spectral analysis showed that the dye X-2 with a large aromatic group formed aggregates in buffer solution displaying very weak fluorescence intensity, and disaggregated in the presence of G-Quadruplex DNAs with large intensity enhancements (up to ~1800 fold). Moreover, X-2 displayed good selectivity to G-Quadruplex DNAs. In contrast, dye X-3 with the smaller aromatic group had much lower fluorescence enhancements and poor selectivity to G-Quadruplex DNAs, suggesting that the suitably sized aromatic ring was essential for the interaction with G-Quadruplex. Further binding studies suggested that X-2 mainly bound on G-quartet surface through end-stacking mode. Cytotoxicity assay showed that both of the two dyes showed good anti-proliferative activities against the cancer cell lines and less cytotoxicity in non-malignant cell lines, which were better than a standard drug 5-fluorouracil. In addition, living cell imaging was also studied and demonstrated the potential applications of the new dye X-2 in bioassays and cell imaging.

A highly sensitive and adjustable colorimetric assay of hydrogen sulfide by signal amplification based on G-quadruplex-Cu2+ peroxidase mimetics.

This work reports the first example of a colorimetric H2S sensor constructed through G-quadruplex-Cu2+ (G4-Cu2+) peroxidase mimetics employing Cu2+ ions and G-rich DNA with signal amplification. In the hydrogen peroxide (H2O2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), the catalytic capacity of Cu2+ can be greatly improved in the presence of 22AG DNA, where 22AG DNA acts as a signal amplifier. However, G4-Cu2+ peroxidase mimetics lose their catalytic abilities after reacting with H2S. This is employed to develop a colorimetric assay of H2S without complex synthesis and instrumentation, with a linear range from 0.01 μM to 150 μM and a detection limit of 7.5 nM. The sensitivity of the sensor can also be adjusted by changing the concentration of Cu2+. Moreover, the developed sensor is successfully applied for the quantitative determination of H2S in human serum samples.

Role of Hfq in Genome Evolution: Instability of G-Quadruplex Sequences in E. coli.

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

ZIF-8-Assisted NaYF4:Yb,Tm@ZnO Converter with Exonuclease III-Powered DNA Walker for Near-Infrared Light Responsive Biosensor.

This work reports a ZIF-8 (ZIF: Zeolitic Imidazolate Framework)-assisted NaYF4:Yb,Tm@ZnO upconverter for the photoelectrochemical (PEC) biosensing of carcinoembryonic antigen (CEA) under near-infrared (NIR) irradiation on a homemade 3D-printed device with DNA walker-based amplification strategy. The composite photosensitive material NaYF4:Yb,Tm@ZnO, as converter to transfer NIR import to photocurrent output, was driven from annealed NaYF4:Yb,Tm@ZIF-8. Yb3+ and Tm3+-codoped NaYF4 (NaYF4:Yb,Tm) converted NIR excitation into UV emission, matching with the absorption of ZnO for in situ excitation to generate the photocurrent. Upon target CEA introduction, the swing arm of DNA walker including the sequence of CEA aptamer carried out the sandwiched bioassembly with CEA capture aptamer on the G-rich anchorage DNA tracks-functionalized magnetic beads. Thereafter, DNA walker was triggered, and the swing arm DNA was captured by the G-rich anchorage DNA according to partly complementary pairing and Exonuclease III (Exo III) consumed anchorage DNA by a burnt-bridge mechanism to go into the next cycle. The released guanine (G) bases from DNA walker enhanced the photocurrent response on a miniature homemade 3D-printed device consisting of the detection cell, dark box, and light platform. Under optimal conditions, NaYF4:Yb,Tm@ZnO-based NIR light-driven PEC biosensor presented high sensitivity and selectivity for CEA sensing with a detection limit of 0.032 ng mL-1. Importantly, our strategy provides a new horizon for the development of NIR-based PEC biosensors in the aspect of developing MOF-derived photoelectric materials, flexible design of a 3D-printed device, and effective signal amplification mode.

An aptamer-based fluorometric zearalenone assay using a lighting-up silver nanocluster probe and catalyzed by a hairpin assembly.

An enzyme-free fluorometric assay is described for the determination of zearalenone (ZEN). The method combines (a) catalyzed hairpin assembly (CHA), (b) ultrahigh fluorescent light-up G-rich DNA sequences in proximity to silver nanoclusters (Ag NCs), and (c) the use of aptamers (Apt). In the presence of ZEN, the inhibit sequence (Inh) is released from the aptamer-trigger sequence (Apt-T) via the binding of ZEN and the aptamer of Apt-T. The free Apt-T acts as a switch that opens the hairpins H1 and H2 to generate H1-H2 complex. The released Apt-T is available to trigger the next round of CHA between H1 and H2. Finally, the hybridization between H1 and the Ag NCs probe (P) causes the G-rich sequence to be in close proximity to the dark Ag NCs encapsulated by P. This leads to highly efficient lighting up of the Ag NCs and the production of amplified fluorescence with excitation/emission peaks at 575/628nm. Under the optimized conditions, a linear correlation was observed with concentrations ranging from 1.3pgmL-1 to 100ngmL-1, and the limit of detection was 0.32pgmL-1 (at S/N = 3). The method was successfully validated by analyzing maize and beer for levels of ZEN after a simple sample preparation procedure. Graphical abstractSchematic of the assay. The inhibit sequence (Inh) is released from aptamer-trigger sequence (Apt-T) via binding of ZEN and aptamer. The free Apt-T triggers catalyzed hairpin assembly (CHA).G-rich DNA is in proximity to silver nanoclusters (Ag NCs) and fluorescence intensity increases to detect ZEN.

A novel electrochemical aptasensor for fumonisin B1 determination using DNA and exonuclease-I as signal amplification strategy

In this work, using DNA and exonuclease-I (Exo-I) as signal amplification strategy, a novel and facile electrochemical aptasensor was constructed for fumonisin B1 (FB1) detection. The G-rich complementary DNA (cDNA) was immobilized onto the electrode surface. Then, aptamer of FB1 was hybridized with cDNA to form double-stranded DNA. In the absence of FB1, double-stranded DNA and G-rich cDNA on the electrode surface promoted effectively methylene blue (MB) enrichment and amplified the initial electrochemical response. In the presence of FB1, the combination of aptamer and FB1 led to the release of aptamer from the electrode surface and the expose of 3′ end of single-stranded cDNA. When Exo-I was added onto the electrode surface, the single-stranded cDNA was degraded in the 3′–5′ direction. The decrease of double-stranded DNA and G-rich cDNA resulted in the less access of MB to the electrode surface, which decreased the electrochemical signal. The experimental conditions including incubation time of FB1, the amount of Exo-I and incubation time of Exo-I were optimized. Under the optimal conditions, the linear relationship between the change of peak current and the logarithmic concentration of FB1 was observed in the range of 1.0 × 10−3–1000 ng mL−1 with a low limit of detection of 0.15 pg mL−1. The experimental results showed that the prepared aptasensor had acceptable specificity, reproducibility, repeatability and stability. Therefore, this proposed aptasensor has a potential application in the food safety detection.

Frontispiece: Targeting G‐Rich DNA Structures with Photoreactive Bis‐Cyclometallated Iridium(III) Complexes

G-rich DNA structures are of great importance, notably in the telomeric region where G-quadruplex DNA plays a key role in the development of cancer. In their Full Paper on page 12730 ff., E. Defrancq, B. Elias et al., report photo-oxidant iridium(III) complexes that can target these DNA structures. As shown on this artwork, the iridium complex is able to trigger an electron transfer with guanine upon light irradiation, with the consequences to increase the prevalence of 8-oxo-dG.

Efficient DNA-Catalyzed Porphyrin Metalation for Fluorescent Ratiometric Pb2+ Detection.

Developing biosensors for Pb2+ is an important analytical topic. DNA-based Pb2+ sensors have been designed mainly based on RNA-cleaving DNAzymes and Pb2+-induced folding of G-quadruplex (G4) DNA. Porphyrin metalation is a key reaction in biology and catalysis. Many enzyme mimics have been developed to catalyze this reaction, and some metalation DNAzymes were reported with a G4 structure. Inspired by the excellent G4 binding properties of certain divalent metal ions, we herein screened a few metals and G-rich DNA sequences. The metalation activity of a DNA named T30695 (sequence: (G3T)4) was significantly accelerated by Pb2+. The reaction of Cu2+ insertion into the mesoporphyrin IX had a kcat of 0.89 min-1 and a Km of 9.8 μM, representing a catalytic efficiency similar to that of human ferrochelatase. The reason for the acceleration was attributed to Pb2+ binding of the G4 DNA and the catalytic activity of the large Pb2+ ion for this reaction. A ratiometric sensor for Pb2+ was developed by inserting Zn2+ with a detection limit of 23.5 nM Pb2+. This work has established a new DNA-based reaction that can be used for Pb2+ detection, and it also provides a highly efficient new DNAzyme for porphyrin metalation, which might be used for signal production for other biosensors.

Target-inspired Pb2+-dependent DNAzyme for ultrasensitive electrochemical sensor based on MoS2-AuPt nanocomposites and hemin/G-quadruplex DNAzyme as signal amplifier

In this work, a novel Pb2+ electrochemical DNAzyme sensor was developed for ultrasensitive detection of lead ions (Pb2+) in water environment by coupling with the MoS2–AuPt nanomaterials and hemin/G-quadruplex DNAzyme, which acting as the electrocatalytic signal tag. Streptavidin (SA) modified tin dioxide-functionalized reduced graphene oxide (rGO-SnO2) /gold nanoparticles (AuNPs) served as a sensor platform for enhancing conductivity and immobilizing more Pb2+-specific DNAzyme. In the presence of Pb2+, the Pb2+-dependent DNAzyme specifically reacted with Pb2+, cleaving the substrate strand (SS) into two free fragment and releasing the biotin-modified enzyme strand (Bio-ES) on the electrode. Connecting MoS2–AuPt nanocomposites labeled with G-rich DNA (G-DNA) strand and exposure of Bio-ES through the Helper DNA, as well as adding hemin to form a hemin/G-quadruplex, the biosensor achieved signal amplification. Chronoamperometry was used to record the current signal, which was primarily derived from the cocatalysis reduction of H2O2 by MoS2–AuPt nanocomposites and the hemin/G-quadruplex. Under optimal conditions, the designed biosensor exhibited sensitive detection of Pb2+ from 0.1 pg mL−1 to 1000 ng mL−1, with a lower detection limit of 38 fg mL−1 (based on 3σ). This proposed biosensor is ultrasensitive and specific, representing a potential application for the detection of Pb2+ in a water environment.

G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications.

Guanine (G)-quadruplexes (G4s) are unique nucleic acid structures that are formed by stacked G-tetrads in G-rich DNA or RNA sequences. G4s have been reported to play significant roles in various cellular events in both macro- and micro-organisms. The identification and characterization of G4s can help to understand their different biological roles and potential applications in diagnosis and therapy. In addition to biophysical and biochemical methods to interrogate G4 formation, G4 fluorescent turn-on ligands can be used to target and visualize G4 formation both in vitro and in cells. Here, we review several representative classes of G4 fluorescent turn-on ligands in terms of their interaction mechanism and application perspectives. Interestingly, G4 structures are commonly identified in DNA and RNA aptamers against targets that include proteins and small molecules, which can be utilized as G4 tools for diverse applications. We therefore also summarize the recent development of G4-containing aptamers and highlight their applications in biosensing, bioimaging, and therapy. Moreover, we discuss the current challenges and future perspectives of G4 fluorescent turn-on ligands and G4-containing aptamers.

Identification of LARK as a novel and conserved G-quadruplex binding protein in invertebrates and vertebrates.

Double-stranded DNAs are usually present in the form of linear B-form double-helix with the base pairs of adenine (A) and thymine (T) or cytosine (C) and guanine (G), but G-rich DNA can form four-stranded G-quadruplex (G4) structures, which plays important roles in transcription, replication, translation and protection of telomeres. In this study, a RNA recognition motif (RRM)-containing protein, BmLARK, was identified and demonstrated to bind G4 structures in the promoters of a transcription factor BmPOUM2 and other three unidentified genes of Bombyx mori, as well as three well-defined G4 structures in the human genes. Homologous LARKs from Bombyx mori, Drosophila melanogaster, Mus musculus and Homo sapiens bound G4 structures in BmPOUM2 and other genes in B. mori and H. sapiens. Upon binding, LARK facilitated the formation and stability of the G4 structure, enhancing the transcription of target genes. The G4 structure was visualized in vivo in cells and testis from invertebrate B. mori and vertebrate Chinese hamster ovary (CHO) cells. The results of this study strongly suggest that LARK is a novel and conserved G4-binding protein and that the G4 structure may have developed into an elaborate epigenetic mechanism of gene transcription regulation during evolution.

Design, synthesis of 4,5-diazafluorene derivatives and their anticancer activity via targeting telomeric DNA G-quadruplex

In our work, 19 novel 4,5-diazafluorene derivatives (11a-d, 12a-d, 13a-d, 14a-c, 15c, 16a-c) bearing a 1,3-disubstituted pyrazol/thioxothiazolidinone or thioxothiazolidinone-oxadiazole moieties were designed, synthesized, preliminarily explored for their antitumor activities and in vitro mechanism. All compounds showed different values of antiproliferative activity against A549, AGS, HepG2 and MCF-7 cell lines through CCK-8. Especially, the compound 14c exhibited the strongest activity and best selectivity against A549 cells with an IC50 1.13 μM and an SI value of 7.01 relative to MRC-5 cells, which was better than cisplatin (SI = 1.80) as a positive control. Experimental results at extracellular level demonstrated that compounds 14a-c could strongly interact with the G-quadruplex(es) formed in a 26 nt telomeric G-rich DNA, in particular, the 14c exhibits quite strong binding affinity with an association equilibrium constant (KA) of 7.04(±0.16) × 107 M−1 and more than 1000-fold specificity to G4-DNA over ds-DNA and Mut-DNA at the compound/G4-DNA ratio of 1:1. Further trap assay ascertained that compounds 14a-c owned strong inhibitory ability of telomerase activity in A549 cells, suggesting that these compounds have great possibility to target telomeric G-quadruplexes and consequently indirectly inhibit the telomerase activity. In addition, it is worthy of note that the remarkable inhibitory effects of 14a-c on the mobility of tested cancer cells were observed by wound healing assays. Furthermore, molecular docking and UV–Vis spectral results unclose the rationale for the interaction of compounds with such G-quadruplex(es). These results indicate that the growth and metastasis inhibition of cancer cells mediated by these 4,5-diazafluorene derivatives possibly result from their interaction with telomeric G-quadruplexes, suggesting that 4,5-diazafluorene derivatives, especially 14c, possess potential as anticancer drugs.

Aptamer-based Targeted Delivery of a G-quadruplex Ligand in Cervical Cancer Cells

AS1411 is a G-rich DNA oligonucleotide that functions as an aptamer of the protein nucleolin, found at high levels on the surface of cancer cells but not on the surface of normal cells. Herein, we have studied AS1411 as a supramolecular carrier for the delivery of an acridine-based G-quadruplex ligand, C8, to HeLa cancer cells. Two AS1411 derivatives, LNA-AS1411 and U-AS1411, were also tested, in an attempt to compare AS1411 pharmacological properties. The results showed that AS1411-C8 complexation was made with great binding strength and that it lowered the ligand’s cytotoxicity towards non-malignant cells. This effect was suggested to be due to a decreased internalization of the complexed versus free C8 as shown by flow cytometry. The AS1411 derivatives, despite forming a stable complex with C8, lacked the necessary tumour-selective behaviour. The binding of C8 to AS1411 G-quadruplex structure did not negatively affect the recognition of nucleolin by the aptamer. The AS1411-C8 repressed c-MYC expression at the transcriptional level, possibly due to C8 ability to stabilize the c-MYC promoter G-quadruplexes. Overall, this study demonstrates the usefulness of AS1411 as a supramolecular carrier of the G-quadruplex binder C8 and the potential of using its tumour-selective properties for the delivery of ligands for cancer therapy.

Label-free Pb2+ detection on the layer-by-layer platform using real-time dual polarization interferometry

Contamination of Lead ion (Pb2+) poses a serious threat to human health and the environment. Developing sensitive and reliable Pb2+ biosensor is essential and significant for prevention and control of the water pollution. In this work, we utilized the direct conformational signal changes in specific interaction process between Pb2+ and guanine-rich (G-rich) oligonucleotides to establish a sensing system. The binding of Pb2+ and G-rich oligonucleotides results in conformational transition from single strand to G-quadruplex, which was recorded real-time by dual polarization interferometry (DPI). The sensing platform was established via layer-by-layer assembly strategy that employing polyethyleneimine and ploythymine as media layers to link the G-rich DNA on the DPI chip, which is simple, facile and without need of synthesizing sophisticated nanomaterial. On the basis of above sensing platform and quantifiable conformational changes, a simple, sensitive, specific and label-free Pb2+ biosensor was established. This research provides us a simple and sensitive method for detection of ions, also encourages us to combine surface technique to biosensing application and explore the relationship between structures and functions.

Telomere DNA G-quadruplex folding within actively extending human telomerase

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase-DNA complex. Addition of the telomerase processivity factor POT1-TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1-TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

Mammalian CST averts replication failure by preventing G-quadruplex accumulation.

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization, even when ATR/ATM pathways were inhibited. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.

Novel strategy to improve the sensing performances of split ATP aptamer based fluorescent indicator displacement assay through enhanced molecular recognition

Split aptamer strategy was often used to improve the sensitivity of aptasensor. However, traditional split aptamer strategy can not be directly used to improve the label-free aptamer based Thioflavin T (ThT) displacement assay for ATP because the split ATP aptamer display much lower enhancement effects on the fluorescence of ThT than intact aptamer. In order to address this issue, this is the first report using G-rich DNA sequence to enhance the affinity of the two split ATP aptamer halves to ThT and offer lower limit of detection (LOD), wider linear range and higher selectivity through the enhanced molecular recognition. Compared to the intact aptamer/ThT complex, the ensemble of two G-rich split ATP aptamer fragments/ThT are higher fluorescent. Consequently, G-rich sequences would improve the fluorescent signal and thus the sensing performance of the proposed assay. In the optimized conditions, the LOD of the proposed fluorescent ATP aptasensor is 2 nM, which is lower than the reported ThT/ATP aptamer based methods. Additionally, our aptasensor has a wider dynamic linear range (0.1 μM - 120 μM) and higher selectivity. The proposed aptasensor has been successfully applied to detect ATP in 15% human serum. More importantly, the current study not only provides a novel method for ATP assay but also presents a way to construct a label-free split aptamer based fluorescent sensor for other species where aptamer can be generated.

High affinity of AS1411 toward copper; its application in a sensitive aptasensor for copper detection

AS1411 is a 26-mer G-rich DNA aptamer which has been broadly employed in the field of targeted drug delivery, due to its capability to bind to nucleolin protein on the surface of cancer cells. In this work, it has been shown for the first time that in addition to nucleolin, AS1411 aptamer could bind to copper ions (Cu2+) with high affinity and selectivity, affecting AS1411 usage in drug delivery systems as a targeting agent. In this study, besides the evaluations of the affinity of AS1411 to different ions and the impact of Cu2+ on targeted drug delivery employed AS1411 aptamer as a targeting agent, a simple and ultra-sensitive fluorescent aptasensor was fabricated for Cu2+ detection through applying AS1411, its complementary strand and magnetic beads coated with streptavidin. Gel Red (GR) was also used as a fluorescent dye. The fabricated aptasensor offered the possibility of condensing samples with different volumes. The detection limit of the sensor was 0.01 μM towards Cu2+ in serum samples. The efficacy of this sensor was further confirmed by comparing Cu2+ levels in serums of healthy people with patients suffering from Wilson's disease, Alzheimer's disease and Diabetes Mellitus using the proposed sensing platform.

Fluorometric determination of the activity of the biomarker terminal deoxynucleotidyl transferase via the enhancement of the fluorescence of silver nanoclusters by in-situ grown DNA tails.

The activity of terminal deoxynucleotidyl transferase (TdTase) is a biomarker for routine diagnosis of acute leukemia. A method has been developed for the determination of TdTase activity. It is based on the use of silver nanoclusters (AgNCs) whose yellow fluorescence is enhanced by an in-situ grown DNA tail of TdTase-polymerized and guanine-rich DNA at the 3' end of a hairpin DNA. The fluorescence, best measured at excitation/emission peaks of 530/585nm, increases linearly in the 1 to 35mUmL-1 TdTase activity range. The detection limit is 0.8mUmL-1. The method is cost-efficient, selective and convenient. It integrates enhancement of the fluorescence of AgNCs and target recognition into a single process. Graphical abstract Schematic presentation of a method for determination of TdTase activity. It is based on AgNCs fluorescence enhanced by in-situ grown TdTase-polymerized G-rich DNA tail. The method integrates AgNCs fluorescence enhancement and the target recognition into a single process.

Identification of G-Quadruplex-Binding Inhibitors of Myc Expression through Affinity Selection–Mass Spectrometry

The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection–mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.