G-quadruplex Sequences Research Articles

In vivo self-degradable graphene nanomedicine operated by DNAzyme and photo-switch for controlled anticancer therapy

Although graphene oxide (GO) possesses many beneficial functionalities for biomedical usage as itself, modification of GO surface with several polymers or protein is inevitable for in vivo applications; however, such modification limits the degradability of GO due to the steric hindrance. In that context, designing of a surface modified GO carrier that is going to be degraded after its biological function (i.e., drug delivery) is highly desired, especially at complex in vivo level. Herein, we design an unprecedented “catalytic GO nanomedicine” by applying the catalytic DNA, achieving self-degradation of GO in systemic level in the body after the therapy following surface modification. Once the catalytic GO nanomedicines are taken up by mucin1 (MUC1) aptamer-facilitated endocytosis, a photo-switch triggers the release of doxorubicin from the DNA. The single stranded G-quadruplex sequence on the surface of GO forms a quartet structure and becomes DNAzyme by binding with hemin on the GO surface, exhibiting peroxidase effect. Due to the high H2O2 concentration in cancer cells, the catalytic GO nanomedicine generates sufficient amount of strong oxidant, hypochlorous acid (HOCl), inducing GO degradation into small fragments for potential clearance. We demonstrate the potential of our catalytic GO nanomedicine for both therapy and degradation at cellular and complex in vivo environment.

Polymerase chain reaction-based ultrasensitive detection of HBV DNA via G-quadruplex selective iridium(III) complex luminescent probe

Polymerase chain reaction (PCR) is the gold standard for low-abundant DNA detection. Here, to expand the application of PCR with novel detecting methods, we developed a label-free fluorescent sensor for ultrasensitive and one-step detection of hepatitis B virus (HBV) DNA using the G-quadruplex selective iridium(III) complex luminescent probe. By using HBV DNA as the template with two hairpin structure primers that contained oxyethylene glycol tethers, PCR amplification occurred and generated numbers of specific PCR products with free G-quadruplex sequences at both ends. Such free G-quadruplex sequences can change into G-quadruplex structure with the help of K+, resulting in a strong luminescence intensity upon their binding with the G-quadruplex selective iridium(III) complex. The luminescence intensity increase was proportional to the concentration of PCR products, and indirectly related with HBV DNA concentration. Moreover, the utilization of the iridium(III) complex effectively improved the specificity of the sensor, while PCR paved the way for the ultrasensitive detection of DNA in the linear range of 3.0 fM to 800 pM, with a detection limit of 1.6 fM. Notably, this assay was successfully used to detect HBV DNA in normal and patient serum samples, indicating a potential application for biomolecular analysis.

Highly Sensitive MicroRNA Detection by Coupling Nicking-Enhanced Rolling Circle Amplification with MoS2 Quantum Dots.

In this work, a label-free and highly sensitive fluorescence assay was constructed for microRNA detection. Nicking-enhanced rolling circle amplification (RCA) induced by G-quadruplex formation is coupled with inner filter effect (IFE)-based quenching effects of MoS2 quantum dots (MoS2 QDs). The padlock probe contains a recognition sequence to target microRNA and an accessible nicking site. The padlock probe is cyclized upon hybridization with target microRNA. Sequentially, amplification initiates a production of a long-concatenated sequence of circular probes. Abundant G-quadruplex sequences are produced via the nicking process and then used as the trigger to initiate the next RCA. In the presence of hemin, numerous hemin/G-quadruplex DNAzymes are formed, which catalyze the oxidation of o-phenylenediamine (OPD) into the colored product 2,3-diaminophenazine, resulting in quenching of the fluorescence of MoS2 QDs. This sensing strategy enables detection of microRNA let-7a with high selectivity and a detection limit of 4.6 fM. The as-prepared sensor was applied for detecting microRNA let-7a in dilute human serum samples and achieved a satisfactory recovery rate, demonstrating its potential in clinic diagnosis of microRNA-associated disease and biochemical research.

One-step G-quadruplex-based fluorescence resonance energy transfer sensing method for ratiometric detection of uracil-DNA glycosylase activity

Uracil-DNA glycosylase (UDG) is a crucial enzyme in base excision repair (BER) pathway. It can repair the uracil-induced DNA lesions and maintain the integrity of genome. In this paper, we developed a facile and ratiometric strategy for UDG activity detection using fluorescence resonance energy transfer (FRET). One double-stranded DNA (dsDNA) substrate consisting of strand 1 (dual-fluorescent dye-modified G-quadruplex sequence single-stranded DNA (ssDNA)), carboxyfluorescein (FAM) acted as donor and tetramethylrhodamine (TAMRA) as acceptor) and strand 2 (the complementary sequence of strand 1 containing three mismatched bases and three uracil bases) was introduced. When the UDG-catalyzed uracil is removed from dsDNA, the thermo-stability of dsDNA is decreased and the dual-fluorescent dye-modified G-quadruplex sequence ssDNA is released. Then, the ssDNA transforms into a G-quadruplex comformation, which brings the labeled FAM and TAMRA into close proximity, resulting in a strong FRET signal. In the absence of UDG, the relatively stable dsDNA separates the labeled FAM and TAMRA, giving a weak FRET signal. Thus, by measuring the system fluorescence intensity and exploiting FRET signal difference, UDG activity can be detected in a simple process. The detection limit is 0.087 U/mL without requiring additional signal amplification process. Besides, our developed strategy can also be used for screening the UDG inhibitors in a ratiometric fluorescence detection way.

G-quadruplex based biosensor: A potential tool for SARS-CoV-2 detection

G-quadruplex is a non-canonical nucleic acid structure formed by the folding of guanine rich DNA or RNA. The conformation and function of G-quadruplex are determined by a number of factors, including the number and polarity of nucleotide strands, the type of cations and the binding targets. Recent studies led to the discovery of additional advantageous attributes of G-quadruplex with the potential to be used in novel biosensors, such as improved ligand binding and unique folding properties. G-quadruplex based biosensor can detect various substances, such as metal ions, organic macromolecules, proteins and nucleic acids with improved affinity and specificity compared to standard biosensors. The recently developed G-quadruplex based biosensors include electrochemical and optical biosensors. A novel G-quadruplex based biosensors also show better performance and broader applications in the detection of a wide spectrum of pathogens, including SARS-CoV-2, the causative agent of COVID-19 disease. This review highlights the latest developments in the field of G-quadruplex based biosensors, with particular focus on the G-quadruplex sequences and recent applications and the potential of G-quadruplex based biosensors in SARS-CoV-2 detection.

Facile strategy to enhance the specificity and sensitivity of hairpin molecular devices for detecting pax-5a gene by an integration probe and the specific function of exonuclease Ⅲ

A label-free DNA sensor is proposed to detect the acute leukemia gene pax-5a, in which the system only relies on a functional hairpin probe (HP) containing a G-quadruplex sequence and the cleavage of exonuclease Ⅲ (Exo Ⅲ). Non-single-stranded fragments in the HP structure can also be digested, including double-stranded stems and G-quadruplex fragments, by Exo Ⅲ at a certain concentration. In the presence of pax-5a, the HP sequence can be cyclically opened through the shearing action of Exo Ⅲ, causing a large number of G-quadruplex fragments to be released. In the absence of pax-5a, the G-quadruplex fragment is digested by Exo Ⅲ. The G-quadruplex-hemin DNAzyme can catalyze the oxidation of 3, 3′, 5, 5′-tetramethylbenzidine by H2O2 to generate a detectable visible absorption signal, thereby achieving facile and visual detection of acute leukemia gene pax-5a. The detection limit of the label-free DNA sensor that we proposed is as low as 25 fM in the pax-5a concentration range of 25 fM to 25 nM. In addition, the sensor has good discrimination ability of non-target genes and excellent detection ability in complex matrices. Therefore, the DNA sensor shows great potential in biological analysis and clinical diagnosis.

FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences.

The sorting of RNA molecules to subcellular locations facilitates the activity of spatially restricted processes. We have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these transcripts contain an enrichment of G-quadruplex sequences in their 3' UTRs, suggesting that FMRP recognizes them to promote RNA localization. We observed similar results in neurons derived from Fragile X Syndrome patients. We identified the RGG domain of FMRP as important for binding G-quadruplexes and the transport of G-quadruplex-containing transcripts. Finally, we found that the translation and localization targets of FMRP were distinct and that an FMRP mutant that is unable to bind ribosomes still promoted localization of G-quadruplex-containing messages. This suggests that these two regulatory modes of FMRP may be functionally separated. These results provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

Functional DNA Structures and Their Biomedical Applications

Since the discovery of the double-helix structure in 1953, nucleic acids have been developed from natural genetic codes into functional building blocks in a wide range of biotechnology and material...

Photoactivated semi-synthetic derivative of osajin selectively interacts with G-quadruplex DNA

Natural and synthetic small molecules targeting G-quadruplex are currently being studied. These peculiar DNA arrangements arise in guanine-rich sequences located in telomeres, oncogene promoters and in several viruses. Two semi-synthetic derivatives of osajin, a natural isoflavone from Maclura pomifera, were prepared and screened for their binding affinity towards G-quadruplex and double stranded DNA (dsDNA) using electrospray mass spectrometry (ESI-MS). Although an overall decrease in affinity for DNA was observed, one of the derivatives showed improved selectivity for G-quadruplex over dsDNA, retaining structure stabilization properties. Binding motif was studied by collision-induced dissociation (CID) assays and molecular docking, that suggested stacking as the preferential interaction pattern. Moreover, the compound selectively induced DNA damage on the G-quadruplex sequence upon UV irradiation, due to the presence of the photoreactive aryl tosylate group.

Potential G-Quadruplex Forming Sequences and N6-Methyladenosine Colocalize at Human Pre-mRNA Intron Splice Sites.

Maturation of mRNA in humans involves modifying the 5′ and 3′ ends, splicing introns, and installing epitranscriptomic modifications that are essential for mRNA biogenesis. With respect to epitranscriptomic modifications, they are usually installed in specific consensus motifs, although not all sequences are modified suggesting a secondary structural component to site selection. Using bioinformatic analysis of published data, we identify in human mature-mRNA that potential RNA G-quadruplex (rG4) sequences colocalize with the epitranscriptomic modifications N6-methyladenosine (m6A), pseudouridine (Ψ), and inosine (I). Using the only available pre-mRNA data sets from the literature, we demonstrate colocalization of potential rG4s and m6A was greatest overall and occurred in introns near 5′ and 3′ splice sites. The loop lengths and sequence context of the m6A-bearing potential rG4s exhibited short loops most commonly comprised of single A nucleotides. This observation is consistent with a literature report of intronic m6A found in SAG (S = C or G) consensus motifs that are also recognized by splicing factors. The localization of m6A and potential rG4s in pre-mRNA at intron splice junctions suggests that these features could function together in alternative splicing. A similar analysis for potential rG4s around sites of Ψ installation or A-to-I editing in mRNA also found a colocalization; however, the frequency was less than that observed with m6A. These bioinformatic analyses guide a discussion of future experiments to understand how noncanonical rG4 structures may collaborate with epitranscriptomic modifications in the human cellular context to impact cellular phenotype.

The Exploration of a New Stable G-Triplex DNA and its Novel Function in Electrochemical Biosensing

A G-triplex, a new kind of DNA structure, has been identified as an intermediate in the folding of G-quadruplexes. However, the studies on G-triplexes are still very limited, and the functions and applications of G-triplexes are further to be developed. In this paper, a new G-triplex sequence (5'-CTGGGAGGGAGGGA-3', G3), obtained by truncating four bases (GGGA) from the 3' end of an 18-base G-quadruplex sequence (G4), was found to significantly decrease the diffusion current of methylene blue (MB). In particular, we proved that a) MB stabilized the structure of G3 and increased the Tm of G3 considerably based on circular dichroism; and b) MB formed a 1:1 non-covalent complex with G3 based on electrospray ionization mass spectrometry. Moreover, molecular dynamics simulations established reliable speculation in the folding topology of G3 and interaction sites between G3 and MB. Based on the strong affinity of G3 with MB, we further developed a novel function of G3 as an electrochemical signal read-out and applied it in the fabrication of a sensitive homogeneous electrochemical aptasensor for cocaine. The features we observed in the G3/MB complex will serve as a new inspiring guideline for developing functional short G-rich ligands.

Luminescent DNAzyme and universal blocking linker Super Polymerase Chain Reaction visual biosensor for the detection of Salmonella

Here, we constructed a fast, universal, “turn-on” biosensor for the highly sensitive visual detection of Salmonella based on luminescent DNAzyme and a universal blocking linker Super Polymerase Chain Reaction (S-PCR). The primer in this biosensor was specially designed. The G-quadruplex sequence is attached to the 5’ end of the primer by a blocking linker and is blocked in the stem part. The S-PCR amplification releases the G-quadruplex, which is incubated with hemin to form DNAzyme to exert peroxide-like activity. The visible colored products are generated by the addition of 3,3′,5,5′-Tetramethylbenzidine. Detection sensitivity is high and, even when the concentration of the Salmonella genome in a sample is as low as 1.5 copies/µL, this color change can be seen with the naked eyes. Moreover, this method is simple and fast, as the S-PCR can be completed in less than 10 min using the newly built equipment and without the need for large instruments.

Multiple signal amplification via coupling DNAzyme with strand displacement reaction for sensitive colorimetric analysis of MUC1

Mucin 1 (MUC1) is abnormally expressed in tumor tissues, and quantitative analysis of MUC1 is thus beneficial to the prevention and early diagnosis of cancer. In this work, by intergrating DNAzyme with strand displacement reaction (SDR), we have fabricated a highly sensitive MUC1 colorimetric assay based on a multiple signal amplification strategy. Specifically, the presence of MUC1 can induce the exposure of the toehold region pre-locked in the aptamer probe (AP) to initiate SDR, liberating the MUC1/AP complex for recycling (cycle I) and forming the dimer-like DNAzyme. The formed DNAzyme can then cyclically cleave the substrate signal probe (cycle II), triggering the formation of G-quadruplex sequence and the new active enzyme sequence that can also cleave the signal probe (cycle III). Such target-induced triple amplification reaction can generate massive G-quadruplex sequences, which leads to a dramatically enhanced colorimetric signal for sensitive detection of MUC1 even at an ultra-low level of 35 pM. The colorimetric strategy also holds a high selectivity to distinguish MUC1 from other interfering proteins and can be successfully applied to the detection of MUC1 in human serum as well as cancer cells, presenting great potential in biosensing and clinical diagnostics.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Integrative analysis reveals RNA G-quadruplexes in UTRs are selectively constrained and enriched for functional associations

G-quadruplex (G4) sequences are abundant in untranslated regions (UTRs) of human messenger RNAs, but their functional importance remains unclear. By integrating multiple sources of genetic and genomic data, we show that putative G-quadruplex forming sequences (pG4) in 5’ and 3’ UTRs are selectively constrained, and enriched for cis-eQTLs and RNA-binding protein (RBP) interactions. Using over 15,000 whole-genome sequences, we find that negative selection acting on central guanines of UTR pG4s is comparable to that of missense variation in protein-coding sequences. At multiple GWAS-implicated SNPs within pG4 UTR sequences, we find robust allelic imbalance in gene expression across diverse tissue contexts in GTEx, suggesting that variants affecting G-quadruplex formation within UTRs may also contribute to phenotypic variation. Our results establish UTR G4s as important cis-regulatory elements and point to a link between disruption of UTR pG4 and disease.

Methods for Identification and Validation of G-Quadruplex Sequences in Legumes.

Among the different secondary structures that DNA can adopt, G-quadruplex is a noncanonical form that has recently started to garner attention about the possible layers of regulation they could introduce in cellular processes. Here, we outline how the presence of G-quadruplexes can be probed in legumes and other plant genomes. This chapter describes various in silico approaches that can be utilized to identify putative G-quadruplex forming sequences (GQSes) and validate their formation through in vitro experimental approaches.

Evolution of specific RNA aptamers via SELEX targeting recombinant human CD36 protein: A candidate therapeutic target in severe malaria

Objective: To isolate and characterize RNA aptamers that are specific to human CD36 protein using systematic evolution of ligands by exponential enrichment (SELEX) technology to identify candidates for adjunct therapy to reverse the binding of Plasmodium-infected erythrocytes. Methods: RNA aptamers were isolated using nitrocellulose membrane-based SELEX and binding analysis was screened using an electrophoretic mobility shift assay and enzyme-linked oligonucleotide assay. Results: Thirteen cycles of nitrocellulose membrane-based SELEX yielded three aptamers (RC60, RC25, RC04) exhibiting high binding against CD36 protein as shown on electrophoretic mobility shift assay. The sequence analysis revealed a G-quadruplex sequence within all the isolated aptamers that might contribute to aptamer binding and thermodynamic stability. The specificity assay further showed that RC60 and RC25 were highly specific to CD36. The competitive inhibition assay demonstrated that RC60 and RC25 shared a similar binding epitope recognized by mAb FA6-152, a specific monoclonal antibody against CD36. Conclusions: RC60 and RC25 are promising candidates as anti-cytoadherence for severe malaria adjunct therapy.

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.

Profusion of G-quadruplexes on both subunits of metazoan ribosomes.

Mammalian and bird ribosomes are nearly twice the mass of prokaryotic ribosomes in part because of their extraordinarily long rRNA tentacles. Human rRNA tentacles are not fully observable in current three-dimensional structures and their conformations remain to be fully resolved. In previous work we identified sequences that favor G-quadruplexes in silico and in vitro in rRNA tentacles of the human large ribosomal subunit. We demonstrated by experiment that these sequences form G-quadruplexes in vitro. Here, using a more recent motif definition, we report additional G-quadruplex sequences on surfaces of both subunits of the human ribosome. The revised sequence definition reveals expansive arrays of potential G-quadruplex sequences on LSU tentacles. In addition, we demonstrate by a variety of experimental methods that fragments of the small subunit rRNA form G-quadruplexes in vitro. Prior to this report rRNA sequences that form G-quadruplexes were confined to the large ribosomal subunit. Our combined results indicate that the surface of the assembled human ribosome contains numerous sequences capable of forming G-quadruplexes on both ribosomal subunits. The data suggest conversion between duplexes and G-quadruplexes in response to association with proteins, ions, or other RNAs. In some systems it seems likely that the integrated population of RNA G-quadruplexes may be dominated by rRNA, which is the most abundant cellular RNA.

A “signal-on” electrochemical biosensor based on DNAzyme-driven bipedal DNA walkers and TdT-mediated cascade signal amplification strategy

In this work, a dual amplified signal enhancement approach based on coupling deoxyribozyme (DNAzyme)-driven bipedal DNA walkers (BDW) and terminal deoxynucleotidyl transferase (TdT)-mediated DNA elongation signal amplifications has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. In presence of thrombin, the BDW complex, which is comprised from the target thrombin and two DNAzyme-containing probes, can exhibit autonomous cleavage behavior on the surface of the substrate DNA (SD) modified electrode, and remove the cleaved DNA fragment from the electrode surface. Subsequently, the TdT can catalyze the elongation of the SD with free 3′-OH termini and formation of many G-quadruplex sequence replicates with the presence of 2′-deoxyaguanosine-5′-triphosphate (dGTP) and adenosine 5′-triphosphate (dATP) at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a “signal-on” and completely label-free fashion. Under optimized conditions, the response peak current was linear with the concentration of thrombin in the range from 0.5 pM to 100000 pM with detection limit of 0.31 pM. This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers.