G-quadruplex Motifs Research Articles

A dual-app nucleoside probe reports G-quadruplex formation and ligand binding in the long terminal repeat of HIV-1 proviral genome

We have developed a dual-app nucleoside analog, 5-selenophene-modified 2′-deoxyuridine (SedU), to probe the structure and ligand-binding properties of a G-rich segment present in the long terminal repeat (LTR) of the HIV-1 proviral DNA promoter region. The nucleoside probe is made of an environment-responsive fluorophore and X-ray crystallography phasing label (Se atom). SedU incorporated into LTR-IV sequence, fluorescently reports the formation of G-quadruplex (GQ) structure without affecting the native fold. Further, using the environment sensitivity of the probe, a fluorescence assay was designed to estimate the binding affinity of small molecule ligands to the GQ motif. An added feature of this probe system is that it would enable direct correlation of structure and recognition properties in solution and atomic level by using a combination of fluorescence and X-ray crystallography techniques.

G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.

Intercalative methylene blue as an efficient signal molecule of tremella-like Bi2WO6: Toward high performance photoelectrochemical biosensing

The signaling molecule is one of the vital factors that determines the detection mechanism/strategy and the resultant performances of photoelectrochemical (PEC) bioassays. This work describes the exploration of the DNA intercalator, i.e. methylene blue (MB), as an efficient signaling molecule for high performance PEC bioanalysis. The MB can stimulate a much larger photocurrent enhancement for tremella-like Bi2WO6 than the other most often used signaling molecules, hinting its higher competence as a signaling molecule. Exemplified by carcinoembryonic antigen (CEA) as a target, we achieved homogeneous and “signal-on” PEC aptasensing, in which the target (CEA)-aptamer binding inhibited the subsequent rolling circle amplification (RCA) reaction, and thus regulating the release/intercalation of MB (in G-quadruplex motif) for signal stimuli. Different from most of the reported anodic PEC bioanalysis with immobilized biomolecules on photoelectrodes, this homogeneous detection not only avoids the cumbersome and costly immobilization/labelling procedure but also facilitates signal transduction, resulting from the well-preserved accessibility and molecular orientation of the biomolecules in solution. This work features the exploration of a DNA intercalator as a signaling molecule for exquisite anodic PEC bioanalysis, which may spark more interests in the exploration of numerous other DNA intercalators for advanced PEC bioanalysis.

Targeting the KRAS oncogene: Synthesis, physicochemical and biological evaluation of novel G-Quadruplex DNA binders

The oncogene KRAS is involved in the pathogenesis of many tumors such as pancreatic, lung and colorectal cancers, thereby representing a relevant target for the treatment of these diseases. The KRAS P1 promoter contains a nuclease hypersensitive, guanine-rich sequence able to fold into a G-quadruplex motif (G4). The stabilization of this G4 structure by small molecules is emerging as a feasible approach to downregulate KRAS expression. Here, a set of novel stabilizing molecules was identified through a virtual screening campaign on the NMR structure of the 22-mer KRAS G4. The most promising hits were then submitted to structure-activity relationships studies which allowed improving their binding affinity and selectivity over double helix DNA and different G4 topologies. The best derivative (19) underwent fluorescence titration experiments and further computational studies to disclose its binding mechanism to KRAS G4. Finally, biological assays showed that this compound is capable to reduce the viability of colorectal cancer cells in which mutated KRAS acts as a driver oncogene. Thus, 19 might represent the prototype of a new class of drugs for the treatment of tumors that, expressing mutated forms of KRAS, are refractory to current therapeutic regimens.

Genome-wide analysis reveals a regulatory role for G-quadruplexes during Adenovirus multiplication

The G-quadruplex (GQ) motifs have recently been gaining prominence because of their role as gene cis-regulatory elements in a variety of organisms and as potential druggable targets for anti-cancer therapy and ageing. Several studies have demonstrated the existence of GQs in the genomes of emerging and re-emerging human pathogens, such as hepatitis virus, herpesviruses, Ebola virus, Zika virus and Nipah virus. Human Adenovirus (HAdV) exhibits a large number of clinical manifestations especially infecting the children and the immunocompromised patients. Moreover, the HAdV-based vectors have been widely used to deliver foreign DNAs to cells in gene therapy. However, the DNA secondary structural elements in AdV-based vectors could significantly determine the gene delivery efficacy of the vectors. In this study, using a combination of whole genome sequence analysis, biochemical, biophysical and interaction assays, we revealed fifteen putative GQs that are conserved across the different species of HAdV. We further showed that the GQs are embedded in the sequences of essential viral genes, namely E1B, E2B, and L3 genes (among others), which are involved in the early and late stages of the viral life cycle. Notably, Braco-19 (a well-known GQ binding ligand) interacted specifically with the HAdV GQs and increased their stability and further blocked the HAdV multiplication in human cells. Taken together, our data strongly supported the existence of G-quadruplex structures in the HAdV genome that affect the virus multiplication and posit that such structures may influence the efficacy of the gene-delivery vectors or even the HAdV virus life-cycle.

Symmetric and dissymmetric carbohydrate-phenyl ditriazole derivatives as DNA G-quadruplex ligands: Synthesis, biophysical studies and antiproliferative activity.

Here we present a novel G4-binding family of compounds based on a central core of phenyl ditriazole (PDTZ) modified with carbohydrates and phenyl pyrrolidinyl side-chains. Their synthesis was achieved using controlled click chemistry conditions to obtain both, symmetric and dissymmetric carb-PDTZ derivatives without any intermediate protecting steps through an optimized methodology. Binding of the new carb-PDTZ to a variety of G-quadruplex motifs was examined using different biophysical techniques. The symmetric carb-PDTZ derivatives were not able to stabilize G4, but the dissymmetric ones (containing one sugar and one phenyl pyrrolidinyl side-chain) did. Interestingly, the dissymmetric carb-PDTZ derivatives showed much higher G4 vs duplex DNA selectivity than the control compound PDTZ 1, which contains two phenyl pyrrodilinyl side-chains and no carbohydrates. Their potential antitumoral activity was also investigated by in vitro cytotoxicity measurements on different cancerous cell lines. All carb-PDTZ derivatives showed higher IC50 values than the control PDTZ 1, probably due to the lack of compound stability of some derivatives and to lower cellular uptake.

Identification and characterization of two conserved G-quadruplex forming motifs in the Nipah virus genome and their interaction with G-quadruplex specific ligands

The G-quadruplex (GQ) motifs are considered as potential drug-target sites for several human pathogenic viruses such as Zika, Hepatitis, Ebola, and Human Herpesviruses. The recent outbreaks of Nipah virus (NiV) in India, the highly fatal emerging zoonotic virus is a potential threat to global health security as no anti-viral drug or vaccine in currently available. Therefore, here in the present study, we sought to assess the ability of the putative G-quadruplex forming sequences in the NiV genome to form G-quadruplex structures and act as targets for anti-viral compounds. Bioinformatics analysis underpinned by various biophysical and biochemical techniques (such as NMR, CD, EMSA, DMS footprinting assay) confirmed the presence of two highly conserved G-quadruplex forming sequences (HGQs) in the G and L genes of NiV. These genes encode the cell attachment glycoprotein and RNA-dependent RNA polymerase, respectively and are essential for the virus entry and replication within the host cell. It remains possible that stabilization of these HGQs by the known G-quadruplex binding ligands like TMPyP4 and Braco-19 represents a promising strategy to inhibit the expression of the HGQ harboring genes and thereby stop the viral entry and replication inside the host cell. Accordingly, we report for the first time, that HGQs in Nipah virus genome are targets for G-quadruplex specific ligands; therefore, could serve as potential targets for anti-viral therapy.

Characterization of G-quadruplex formation in the ARID1A promoter

As a member of the SWI/SNF family, ARID1A plays an essential role in modulating chromatin structure and gene expression. The tumor suppressive function of ARID1A has been well-defined and its downregulation in cancers is attributed to genomic deletion, DNA methylation and microRNA-mediated inhibition. In this study, we demonstrated that the negative strand of a C-rich region in the upstream vicinity of the human ARID1A transcription start site could form G-quadruplexes. Synthesized oligonucleotides based on the sequence of this region exhibited molar ellipticity at specific wavelengths characteristic of G-quadruplex structures in circular dichroism analyses. The formation of G-quadruplexes by these oligonucleotides were also proved by native polyacrylamide gel electrophoresis, DNA synthesis block assays, immunofluorescent staining and dimethyl sulfate footprinting studies. In reporter assays, mutations of the G-quadruplex forming sequence reduced ARID1A promoter-mediated transcription. Transfection of the oligonucleotide with the full length of G-quadruplex motif region, but not its partial sequences or the mutants, could both promote endogenous ARID1A expression and reduce cell proliferation.

Prion protein transcription is auto-regulated through dynamic interactions with G-quadruplex motifs in its own promoter

Cellular prion protein (PrP) misfolds into an aberrant and infectious scrapie form (PrPSc) that lead to fatal transmissible spongiform encephalopathies (TSEs). Association of prions with G-quadruplex (GQ) forming nucleic acid motifs has been reported, but implications of these interactions remain elusive. Herein, we show that the promoter region of the human prion gene (PRNP) contains two putative GQ motifs (Q1 and Q2) that assume stable, hybrid, intra-molecular quadruplex structures and bind with high affinity to PrP. Here, we investigate the ability of PrP to bind to the quadruplexes in its own promoter. We used a battery of techniques including SPR, NMR, CD, MD simulations and cell culture-based reporter assays. Our results show that PrP auto-regulates its expression by binding and resolving the GQs present in its own promoter. Furthermore, we map this resolvase-like activity to the N-terminal region (residues 23–89) of PrP. Our findings highlight a positive transcriptional-translational feedback regulation of the PRNP gene by PrP through dynamic unwinding of GQs in its promoter. Taken together, our results shed light on a yet unknown mechanism of regulation of the PRNP gene. This work provides the necessary framework for a plethora of studies on understanding the regulation of PrP levels and its implications in prion pathogenesis.

Correction: Copper(ii) l/d-valine-(1,10-phen) complexes target human telomeric G-quadruplex motifs and promote site-specific DNA cleavage and cellular cytotoxicity.

Correction for 'Copper(ii) l/d-valine-(1,10-phen) complexes target human telomeric G-quadruplex motifs and promote site-specific DNA cleavage and cellular cytotoxicity' by Farukh Arjmand et al., Dalton Trans., 2020, 49, 9888-9899, DOI: 10.1039/d0dt01527j.

Theoretical insights into the effect of size and substitution patterns of azobenzene derivatives on the DNA G-quadruplex.

Introducing photoswitches into the DNA G-quadruplex provides excellent opportunities to control folding and unfolding of these assemblies, demonstrating their potential in the development of novel nanodevices with medical and nanotechnology applications. Using a quantum mechanics/molecular mechanics (QM/MM) scheme, we carried out a series of simulations to identify the effect of the size and substitution patterns of three azobenzene derivatives (AZ1, AZ2 and AZ3) on the excitation energies of the two lowest excited states of the smallest photoswitchable G-quadruplex reported to date. We demonstrated that the size and the substitution pattern do not affect the ultrafast cis-trans photoiomerization mechanism of the azobenzene derivatives significantly, in agreement with the experiment. However, molecular dynamics simulations revealed that while AZ2 and AZ3 G-quadruplexes are structurally stable during the simulations, the AZ1 G-quadruplex undergoes larger structural changes and shows two ground state populations that differ in the azobenzene backbone adopting two different conformations. AZ1, with para-para substitution pattern, provides more flexibility to the whole G-quadruplex structure compared to AZ2 and AZ3, and can thus facilitate the photoisomerization reaction between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after trans-cis isomerization occurring in a longer time dynamics, in agreement with the experimental findings. The QM/MM simulations of the absorption spectra indicated that the thermal fluctuation plays a more crucial role in the main absorption band of the azobenzene derivatives than the inclusion of the G-quadruplex, implying that the influence of the G-quadruplex environment is minimal. We propose that the latter is attributed to the position of the azobenzene linkers in the G-quadruplexes, i.e. the edgewise loops containing the azobenzene moieties that are located above the G-quartets, not being fully embedded inside or involved in the stacked structure. Our theoretical findings provide support to a recent study of the photoresponsive formation of photoswitchable G-quadruplex motifs.

Telomere repeat–binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters

The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex–forming DNA sequences. Furthermore, we validated TRF2 occupancy at several promoter G-quadruplex motifs, which did adopt quadruplex forms in solution. TRF2 binding altered expression and the epigenetic state of several target promoters, indicated by histone modifications resulting in transcriptional repression of eight of nine genes investigated here. Furthermore, TRF2 occupancy and target gene expression were also sensitive to the well-known intracellular G-quadruplex–binding ligand 360A. Together, these results reveal an extensive genome-wide association of TRF2 outside telomeres and that it regulates gene expression in a G-quadruplex–dependent fashion.

Alternatively spliced variants of the 5’-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif

ABSTRACTThe 5ʹ-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5ʹ-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stem-loop element. Taken together, our study describes the functional relevance of two alternative 5ʹ-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions

Conserved G-Quadruplexes Regulate the Immediate Early Promoters of Human Alphaherpesviruses.

Human Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.

Integrative characterization of G-Quadruplexes in the three-dimensional chromatin structure

ABSTRACTDNA molecules are highly compacted in the eukaryotic nucleus where distal regulatory elements reach their targets through three-dimensional chromosomal interactions. G-quadruplexes, stable four-stranded non-canonical DNA structures, can change local chromatin organization through the exclusion of nucleosomes. However, the relationship between G-quadruplexes and higher-order genome organization remains unknown. Here, we found that G-quadruplexes are significantly enriched at boundaries of topological associated domains (TADs). Architectural protein occupancy, which plays critical roles in the formation of TADs, was highly correlated with the content of G-quadruplexes at TAD boundaries. Moreover, adjacent boundaries containing G-quadruplexes frequently interacted with each other because of the high enrichment of architectural protein binding sites. Similar to CCCTC-binding factor (CTCF) binding sites, G-quadruplexes also showed strong insulation ability in the separation of adjacent regions. Additionally, the insulation ability of CTCF binding sites and TAD boundaries was significantly reinforced by G-quadruplexes. Furthermore, G-quadruplex motifs on different strands were associated with the orientation of CTCF binding sites. These findings suggest a potential role for G-quadruplexes in loop extrusion. The enrichment of transcription factor binding sites (TFBSs) around regulatory elements containing G-quadruplexes led to frequent interactions between regulatory elements containing G-quadruplexes. Intriguingly, more than 99% of G-quadruplexes overlapped with TFBSs. The binding sites of CTCF and cohesin proteins were preferentially located surrounding G-quadruplexes. Accordingly, we proposed a new mechanism of long-distance gene regulation in which G-quadruplexes are involved in distal interactions between enhancers and promoters.

Binding between G Quadruplexes at the Homodimer Interface of the Corn RNA Aptamer Strongly Activates Thioflavin T Fluorescence

Thioflavin T (ThT) is widely used for the detection of amyloids. Many unrelated DNAs and RNAs that contain G-quadruplex motifs also bind ThT and strongly activate its fluorescence. To elucidate the structural basis of ThT binding to G quadruplexes and its fluorescence turn-on, we determined its co-crystal structure with the homodimeric RNA Corn, which contains two G quadruplexes. We found that two ThT molecules bind in the dimer interface, constrained by a G quartet from each protomer into a maximally fluorescent planar conformation. The unliganded Corn homodimer crystal structure reveals a collapsed fluorophore-binding site. In solution, Corn must fluctuate between this and an open, binding-competent conformation. A co-crystal structure with another benzothiazole derivate, thiazole orange (TO), also shows binding at the Corn homodimer interface. As the bound ThT and TO make no interactions with the RNA backbone, their Corn co-crystal structures likely explain their fluorescence activationupon sequence-independent DNA and RNA G-quadruplex binding.

Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets

G-quadruplex structure forming motifs are among the most studied evolutionarily conserved drug targets that are present throughout the genome of different organisms and susceptible to influencing various biological processes. Here we report highly conserved potential G-quadruplex motifs (PGQs) in three essential genes (espK, espB, and cyp51) among 160 strains of the Mycobacterium tuberculosis genome. Products of these genes are involved in pathways that are responsible for virulence determination of bacteria inside the host cell and its survival by maintaining membrane fluidity. The espK and espB genes are essential players that prevent the formation of mature phagolysosome and antigen presentation by host macrophages. The cyp51 is another PGQ-possessing gene involved in sterol biosynthesis pathway and membrane formation. In the present study, we revealed the formation of stable intramolecular parallel G-quadruplex structures by Mycobacterium PGQs using a combination of techniques (NMR, circular dichroism [CD], and gel electrophoresis). Next, isothermal titration calorimetry (ITC) and CD melting analysis demonstrated that a well-known G-quadruplex ligand, TMPyP4, binds to and stabilizes these PGQ motifs. Finally, polymerase inhibition and qRT-PCR assays highlight the biological relevance of PGQ-possessing genes in this pathogen and demonstrate that G-quadruplexes are potential drug targets for the development of effective anti-tuberculosis therapeutics.

Characterization of highly conserved G-quadruplex motifs as potential drug targets in Streptococcus pneumoniae

Several G-quadruplex forming motifs have been reported to be highly conserved in the regulatory regions of the genome of different organisms and influence various biological processes like DNA replication, recombination and gene expression. Here, we report the highly conserved and three potentially G-quadruplex forming motifs (SP-PGQs) in the essential genes (hsdS, recD, and pmrA) of the Streptococcus pneumoniae genome. These genes were previously observed to play a vital role in providing the virulence to the bacteria, by participating in the host-pathogen interaction, drug-efflux system and recombination- repair system. However, the presence and importance of highly conserved G-quadruplex motifs in these genes have not been previously recognized. We employed the CD spectroscopy, NMR spectroscopy, and electrophoretic mobility shift assay to confirm the adaptation of the G-quadruplex structure by the SP-PGQs. Further, ITC and CD melting analysis revealed the energetically favorable and thermodynamically stable interaction between a candidate G4 binding small molecule TMPyP4 and SP-PGQs. Next, TFP reporter based assay confirmed the regulatory role of SP-PGQs in the expression of PGQ harboring genes. All these experiments together characterized the SP-PGQs as a promising drug target site for combating the Streptococcus pneumoniae infection.

X-Ray Crystallographic Studies of G-Quadruplex Structures.

The application of X-ray crystallographic methods toward a structural understanding of G-quadruplex (G4) motifs at atomic level resolution can provide researchers with exciting opportunities to explore new structural arrangements of putative G4 forming sequences and investigate their recognition by small molecule compounds. The crowded and ordered crystalline environment requires the self-assembly of stable G4 motifs, allowing for an understanding of their inter- and intramolecular interactions in a packed environment, revealing thermodynamically stable topologies. Additionally, crystallographic data derived from these experiments in the form of electron density provides valuable opportunities to visualize various solvent molecules associated with G4s along with the geometries of the metal ions associated within the central channel-elements critical to the understanding G4 stability and topology. Now, with the advent of affordable, commercially sourced and purified synthetic DNA and RNA molecules suitable for immediate crystallization trials, and combined with the availability of specialized and validated crystallization screens, researchers can now undertake in-house crystallization trials without the need for local expertise. When this is combined with access to modern synchrotron platforms that offer complete automation of the data collection process-from the receipt of crystals to delivery of merged and scaled data for the visualization of electron density-the application of X-ray crystallographic techniques is made open to nonspecialist researchers. In this chapter we aim to provide a simple how-to guide to enable the reader to undertake crystallographic experiments involving G4s, encompassing the design of oligonucleotide sequences, fundamentals of the crystallization process and modern strategies used in setting up successful crystallization trials. We will also describe data collection strategies, phasing, electron density visualization, and model building. We will draw on our own experiences in the laboratory and hopefully build an appreciation of the utility of the X-ray crystallographic approaches to investigating G4s.

Prediction of the secondary structure of short DNA aptamers.

Aptamers have a spectrum of applications in biotechnology and drug design, because of the relative simplicity of experimental protocols and advantages of stability and specificity associated with their structural properties. However, to understand the structure-function relationships of aptamers, robust structure modeling tools are necessary. Several such tools have been developed and extensively tested, although most of them target various forms of biological RNA. In this study, we tested the performance of three tools in application to DNA aptamers, since DNA aptamers are the focus of many studies, particularly in drug discovery. We demonstrated that in most cases, the secondary structure of DNA can be reconstructed with acceptable accuracy by at least one of the three tools tested (Mfold, RNAfold, and CentroidFold), although the G-quadruplex motif found in many of the DNA aptamer structures complicates the prediction, as well as the pseudoknot interaction. This problem should be addressed more carefully to improve prediction accuracy.