Guanine-rich DNA Sequences Research Articles

High-Throughput Quantitative Screening of Peroxidase-Mimicking DNAzymes on a Microarray by Using Electrochemical Detection

Some guanine-rich DNA sequences, which are called DNAzymes, can adopt G-quadruplex structures and exhibit peroxidase activity by binding with hemin. Although known DNAzymes show less activity than horseradish peroxidase, they have the potential to be widely used for the detection of target molecules in enzyme-linked immunosorbent assays if sequences that exhibit higher activity can be identified. However, techniques for achieving this have not yet been described. Therefore, we compared the DNAzyme activities of more than 1000 novelistically designed sequences with that of the original DNAzyme by using an electrochemical detection system on a 12K DNA microarray platform. To the best of our knowledge, this is the first description of an array-based assessment of peroxidase activity of G-quadruplex-hemin complexes. By using this novel assay system, more than 200 different mutants were found that had significantly higher activities than the original DNAzyme sequence. This microarray-based DNAzyme evaluation system is useful for identifying highly active new DNAzymes that might have potential as tools for developing DNA-based biosensors with aptamers.

Assaying the binding strength of G-quadruplex ligands using single-molecule TPM experiments

G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na+ or K+). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10mM Na+ solution at 37°C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68±0.01 and 1.63±0.03 (s−1), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu2+-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions.

50 Single-molecule studies of human telomeric G-quadruplexes and the effect of oxidative damage

In the human genome, telomeric DNA has tandem repeats of the sequence 5′-TTAGGG terminating with a 3′ single-stranded overhang of 100–200 bases. These guanine-rich DNA sequences can fold into tetrastranded structures, known as G-quadruplexes. The precise fold of the G-quadruplex structure is dictated by the metal ions present which we studied through the use of the α-hemolysin ion channel. Being electrophoretically driven into the cis side of the α-hemolysin, the hybrid fold (K+) entered the vestibule mouth leading to current blockages for the duration of the time the DNA resided in the vestibule. Due to the polymorphic nature of the hybrid folds, the recorded current signatures could be correlated with the major structural topologies that exist for this fold in solution (e.g. hybrid-1, hybrid-2, and triplex; Mashimo et al., 2010). The hybrid folds were not capable of traversing to the trans side of the nanopore, while the triplex could achieve translocation. The basket fold (Na+) was also able to enter into the vestibule causing current blockages that were indicative of the orientation in which they entered into the vestibule. When the basket fold entered tail first, slow translocation events were observed. In contrast, the propeller fold (∼3.9 nm, 0.05 M K+/5 M Li+) exceeds the protein channel orifice (∼3.0 nm) producing only swift and small disturbances to the open channel current. Secondly, oxidative damage to the telomeric sequence is proposed to contribute to telomere shortening, dysfunction, and cell aging (Epel et al., 2004). Locations of the oxidative damages have different effects on the G-quadruplex folding that produced significant changes in their nanopore behavior. Placement of the guanine oxidation product, 8-oxoguanosine (OG), in a top or bottom tetrad results in destabilization of that layer, whereas the presence of OG in a middle tetrad leads to complete unfolding of the G-quadruplex. These behaviors were determined by their translocation times which correlated with the folding’s free energy supported by NIH GM093099.

DNA-Templated Silver Nanoclusters that Fluoresce upon Hybridization

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences, termed enhancer sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that “lights up” upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. In addition, we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. We have exploited this color shift to directly detect single nucleotide polymorphisms (SNPs). This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

Binding-Induced Fluorescence Turn-On Assay Using Aptamer-Functionalized Silver Nanocluster DNA Probes

We present here a binding-induced fluorescence turn-on assay for protein detection. Key features of this assay include affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters (Ag NCs) using guanine-rich DNA sequences. In an example of an assay for human α-thrombin, two aptamers (Apt15 and Apt29) were used and were modified by including additional sequence elements. A 12-nucleotide (nt) sequence was used to link the first aptamer with a nanocluster nucleation sequence at the 5'-end. The second aptamer was linked through a complementary sequence (12-nt) to a G-rich overhang at the 3'-end. Binding of the two aptamer probes to the target protein initiates hybridization between the complementary linker sequences attached to each aptamer and thereby bring the end of the G-rich overhang to close proximity to Ag NCs, resulting in a significant fluorescence enhancement. With this approach, a detection limit of 1 nM and a linear dynamic range of 5 nM-2 μM were achieved for human α-thrombin. This fluorescence assay is performed in a single tube, and it does not require washing or separation steps. The principle of the binding-induced DNA hybridization and fluorescence enhancement of Ag NCs can be extended to other homogeneous assay applications provided that two appropriate probes are available to bind with the same target molecule.

Abstract 1767: Anthracenyl isoxazole amides (AIMs) as novel agents for brain tumors

Abstract In the United States, the average survival rate for malignant brain tumors is approximately one year. Despite recent advancements in treatment, there remains a need for the development of new therapeutic agents to target and treat malignant brain tumors to increase survival and quality of life. Due to its pivotal role in cancer cell proliferation, telomerase has become an attractive target in the development of new anticancer agents. The enzymatic activity of telomerase is responsible for telomere maintenance and adds the guanine-rich DNA sequence repeats to the 3′ end of DNA strands in the telomere regions. Human telomeric DNA has the potential to adopt G-quartet conformations, which in turn can stack in various arrangements to form G quadruplexes (G4). To this end, molecules that bind telomeric G4 DNA and inhibit telomerase have substantial anticancer potential. The objective of this study was to evaluate the cytotoxicity of a novel series of anthracenyl isoxazole amides (AIMs), capable of selectively binding to G4 telomeric DNA, and determine the ability of the AIMs to penetrate cell membranes and enter the nuclei of cancer cells. These AIMs have demonstrated significant activity against brain tumor cell lines in the DTP NCI-60 cell line panel, and COMPARE analysis indicated that these molecules exerted their cytotoxicity via a unique mechanism. Two AIMs, referred to here as AIM-dimer and AIM-double tail (AIM-dt), showed single digit micromolar IC50 values (4.75 +/− 0.27 and 3.13 +/− 0.45, respectively) by MTT assay when tested in the human glioblastoma cell line SNB-19. As a followup to the cytotoxicity studies, apoptosis was evaluated by flow cytometry (Annexin V). The results indicated that both AIMs induced apoptosis in SNB-19 cells at higher levels than untreated cells after a four-hour treatment. Since the AIMs are known to autofluoresce, confocal microscopy was performed to determine if they were able to penetrate cell membranes of the SNB-19 cells. Contrary to bioavailability calculations made with Symyx Draw v3.1, which suggested that only the AIM-dt had acceptable properties for cell penetration, both compounds were found to enter the cells. Perhaps more intriguing, however, are the results from the laser scanning cytometry (iCys) studies. SNB-19 cells were co-treated with either AIM-dimer or AIM-dt and the nuclear stain propidium iodide. Images taken of each AIM clearly indicated co-localization of the AIM at the nucleus, suggesting that these AIMs are able to penetrate cell membranes and reach the nuclei and therefore have potential to bind DNA. Further studies will be focused on elucidating the mechanism of action of the AIMs as well as confirming their interactions at the level of G4 telomeric DNA. Supported by NIH Grant P20RR017670 (HDB). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1767. doi:1538-7445.AM2012-1767

Stability and Structure of Long Intramolecular G-Quadruplexes

G-quadruplexes are formed from guanine-rich sequences of DNA and RNA. They consist of stacks of square arrangements of guanines called G-quartets. Increasing evidence suggests that these structures are involved in cellular processes such as transcription or translation. Knowing their structure and their stability in vitro should help to predict their formation in vivo and to understand their biological functions. Many studies have been performed on isolated G-quadruplexes, but little attention has been given to their interactions. Here, we present non-denaturing gel electrophoresis, UV melting, and circular dichroism data obtained for long sequences of DNA which are capable of forming two simultaneous G-quadruplexes, namely, d(TG(3)T(3)G(3)T(3)G(3)T(3)G(3)T(n)G(3)T(3)G(3)T(3)G(3)T(3)G(3)T), with n varying from one to seven. These sequences can form up to two separate G-quadruplexes. We also study mutated versions of these sequences designed to form one G-quadruplex at specific positions on the strand. Comparing results from the original sequences and their mutated versions, we show that for the former different folded states coexist: either with six stacked G-quartets or only three, in various combinations. Which ones are favored depends on n. Moreover, for n greater than three, the thermodynamic stability stays constant, contrary to an expected decrease in stability if the six G-quartets were stacked together in a single structure. This result agrees with a beads-on-a-string folding model for long sequences of G-quadruplexes, where two adjacent G-quadruplexes fold independently.

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here, we show that the G-quadruplex interacting drug pyridostatin promoted growth arrest in human cancer cells via inducing replication- and transcription-dependent DNA damage. Chromatin immunoprecipitation sequence (ChIP-Seq) analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage, and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein levels and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.

The Color Switching Behavior of DNA-Templated Silver Nanoclusters

Oligonucleotide-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that have seen applications in cellular imaging and chemical/biological detection. We discovered the red fluorescence emission of DNA/Ag NCs could be enhanced more than 500 fold when these silver nanoclusters were brought close to a guanine-rich DNA sequence (called an enhancer). Based on this finding, we developed a new type of molecular probe termed NanoCluster Beacon (NCB) that fluoresces upon target DNA binding (1). Expanding upon this work, recently we found non-emissive DNA/Ag NCs can light up into three distinct colors (green, yellow, and red) through interactions with three different DNA enhancers respectively (2), enabling the use of NCBs in multiplexed assays. In order to investigate the enhancer-mediated, color switching behavior of DNA/Ag NCs, a toehold DNA displacement technique was used to replace old enhancers with new enhancers in the proximity of nanoclusters at room temperature. Our results showed that the light-up color can change from green to red if the green enhancer is replaced with the red enhancer, but not vice versa. Similar behavior was also seen from yellow to red color switching. This information may be useful in future silver nanocluster probe design. Reference: [1] H.-C. Yeh, J. Sharma, J.J. Han, J.S. Martinez and J.H. Werner, “A DNA-silver nanocluster probe that fluoresces upon hybridization,” Nano Letters 10(8): 3106-3110, 2010. [2] H.-C. Yeh, J. Sharma, J.J. Han, J.S. Martinez and J.H. Werner, “A beacon of light: a new molecular probe for homogeneous detection of nucleic acid targets,” IEEE Nanotechnology Magazine 5(2): 28-33, 2011.

Explaining the varied glycosidic conformational, G-tract length and sequence preferences for anti-parallel G-quadruplexes.

Guanine-rich DNA sequences tend to form four-stranded G-quadruplex structures. Characteristic glycosidic conformational patterns along the G-strands, such as the 5′-syn-anti-syn-anti pattern observed with the Oxytricha nova telomeric G-quadruplexes, have been well documented. However, an explanation for these featured glycosidic patterns has not emerged. This work presents MD simulation and free energetic analyses for simplified two-quartet [d(GG)]4 models and suggests that the four base pair step patterns show quite different relative stabilities: syn-anti > anti-anti > anti-syn > syn-syn. This suggests the following rule: when folding, anti-parallel G-quadruplexes tend to maximize the number of syn-anti steps and avoid the unfavorable anti-syn and syn-syn steps. This rule is consistent with most of the anti-parallel G-quadruplex structures in the Protein Databank (PDB). Structural polymorphisms of G-quadruplexes relate to these glycosidic conformational patterns and the lengths of the G-tracts. The folding topologies of G2- and G4-tracts are not very polymorphic because each strand tends to populate the stable syn-anti repeat. G3-tracts, on the other hand, cannot present this repeating pattern on each G-tract. This leads to smaller energy differences between different geometries and helps explain the extreme structural polymorphism of the human telomeric G-quadruplexes.

Studies of Quadruplex Folding using a New Fluorescent Porphyrin Derivative

Small molecules that bind selectively to guanine-rich telomeric DNA sequences (quadruplex interactive agents; QIAs) can promote folding to a four-stranded quadruplex conformation. Such quadruplexes have been shown to deactivate telomerase, a key activating enzyme in many cancers, making QIAs promising targets for development of potential cancer drugs. Porphyrin-based QIAs offer several desirable properties as they are non-toxic and found in many oxidation-reduction proteins of the cell. In this study we have examined the effects of a new fluorescent pyropheophorbide porphyrin derivative (PPP-D) on the folding of model human telomeric DNA sequences (HT4). PPP-D demonstrates enhanced lipid solubility (log P = 6.80 ± 1.62) over other quadruplexed-DNA binding porphyrins, including meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and N-methyl mesoporphyrin IX (NMM), and can facilitate cellular delivery (log P = 6.80 ± 1.62) through the cell membrane. Unlike TMPyP4 and NMM, PPP-D is non fluorescent in buffer solutions and highly fluorescent in organic solvents and non-polar environments. CD studies suggest that PPP-D successfully aids in the folding of HT4 sequences in addition to stabilizing the quadruplexed DNA conformation. Furthermore, PPP-D demonstrates selectivity for the quadruplexed (q-DNA) over duplexed DNA (d-DNA) conformation. The significance of these results for anti-tumorigenesis will be discussed. Supported by NIH-SCORE grants ISCI-GM093830-01 (AG) and S06-GM076168-01 (LD).

Nanocluster Beacon (NCB): A DNA-Silver Nanocluster Probe that Fluoresces upon Hybridization

Oligonucleotide-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that have seen applications in cellular imaging and chemical/biological detection. Recently we discovered the red fluorescence emission of DNA/Ag NCs could be enhanced more than 500 fold when brought into close proximity to a guanine-rich DNA sequence (Yeh et al., Nano Letters, 10 (8): 3106-3110, 2010). Based on this finding, we developed a new type of molecular probe (termed NanoCluster Beacon, NCB) that fluoresces upon target DNA binding. Compared to molecular beacons, NCBs require only a single labeling step and do not rely on F [[Unable to Display Character: ő]]rster energy transfer as fluorescence switching mechanism. Moreover, there is no need to remove the silver nanocluster precursors (Ag+ and BH4-) used during nanocluster formation, as these are essentially non-fluorescent. Our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers. It is also the first demonstration that a turn-on probe can be made based on fluorescent noble metal nanoclusters.Our initial investigation demonstrated NCB detection of an influenza target with a signal-to-background (S/B) ratio five times better than that of a conventional molecular beacon. Here, we expand upon this work to demonstrate a method of using NCBs to differentiate single-nucleotide variations. Our method discriminates single-nucleotide variants by colorimetric change of the NCB probes rather than fluorescence intensity change. This added dimensionality enables the increase in fluorescence intensity (on/off switching) to quantify the amount of target, whereas the fluorescence color identifies single-nucleotide variants. Samples with single-nucleotide variations can be unambiguously identified on a common gel imager with naked eyes, making this method a reliable and low-cost assay with simple readout format.Ref: H.-C. Yeh et al., “A DNA-silver nanocluster probe that fluoresces upon hybridization,” Nano Letters 10 (8): 3106-3110, 2010.

Kinetically governed polymorphism of d(G 4 T 4 G 3 ) quadruplexes in K + solutions

It has been generally recognized that understanding the molecular basis of some important cellular processes is hampered by the lack of knowledge of forces that drive spontaneous formation/disruption of G-quadruplex structures in guanine-rich DNA sequences. According to numerous biophysical and structural studies G-quadruplexes may occur in the presence of K+ and Na+ ions as polymorphic structures formed in kinetically governed processes. The reported kinetic models suggested to describe this polymorphism should be considered inappropriate since, as a rule, they include bimolecular single-step associations characterized by negative activation energies. In contrast, our approach in studying polymorphic behavior of G-quadruplexes is based on model mechanisms that involve only elementary folding/unfolding transitions and structural conversion steps that are characterized by positive activation energies. Here, we are investigating a complex polymorphism of d(G4T4G3) quadruplexes in K+ solutions. On the basis of DSC, circular dichroism and UV spectroscopy and polyacrylamide gel electrophoresis experiments we propose a kinetic model that successfully describes the observed thermally induced conformational transitions of d(G4T4G3) quadruplexes in terms of single-step reactions that involve besides single strands also one tetramolecular and three bimolecular quadruplex structures.

ChemInform Abstract: Interaction of Metal Complexes with G‐Quadruplex DNA

AbstractReview: 105 refs.

A DNA−Silver Nanocluster Probe That Fluoresces upon Hybridization

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that "lights up" upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

Metallo-Organic G-Quadruplex Ligands in Anticancer Drug Design

Guanine-rich DNA sequences can form planar four-stranded structures via Hoogsteen hydrogen bonds. These sequences in telomeric DNA and some oncogenes have been identified as targets for novel anticancer drugs. Consequently, there is great current interest in developing small molecules that can facilitate the formation of, and stabilize G-quadruplexes as potent antitumor chemotherapeutic agents. Metal complexes with planar geometries and phi-delocalised ligands are emerging as a new class of quadruplex DNA ligand with unique features for optimal binding. This review will summarize the different types of metal-based compounds that target G-quadruplexes and provide useful information on the design of small ligands.

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and with genomic instability. An important example is sequences containing G-rich homopurine-homopyrimidine stretches, for which unusual transcriptional behavior is implicated in regulation of immunogenesis and in other processes such as genomic translocations and telomere function. To elucidate the mechanism of the effect of these sequences on transcription we have studied T7 RNA polymerase transcription of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, length- and supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution conditions, and ribonucleotide substitutions, we conclude that transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation. These structures are likely responsible for transcription-dependent replication blockage by G-rich sequences in vivo.

Interaction of Metal Complexes with G‐Quadruplex DNA

Guanine-rich sequences of DNA can assemble into tetrastranded structures known as G-quadruplexes. It has been suggested that these secondary DNA structures could be involved in the regulation of several key biological processes. In the human genome, guanine-rich sequences with the potential to form G-quadruplexes exist in the telomere as well as in promoter regions of certain oncogenes. The identification of these sequences as novel targets for the development of anticancer drugs has sparked great interest in the design of molecules that can interact with quadruplex DNA. While most reported quadruplex DNA binders are based on purely organic templates, numerous metal complexes have more recently been shown to interact effectively with this DNA secondary structure. This Review provides an overview of the important roles that metal complexes can play as quadruplex DNA binding molecules, highlighting the unique properties metals can confer to these molecules.

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases.

Using principal component analysis to find correlations between loop-related and thermodynamic variables for G-quadruplex-forming sequences

The application of Principal Component Analysis (PCA) is proposed here as a simple means of revealing correlations between thermodynamic variables corresponding to folding equilibria of intramolecular G-quadruplexes and Watson–Crick duplexes, and the length of loops in the corresponding guanine-rich DNA sequences. To this end, two previously studied data sets were analyzed (Arora and Maiti, J . Phys. Chem. B. 2009 and Kumar and Maiti, Nucleic Acids. Res. 2008). All of the sequences considered shared the common structure 5’- GGG - loop1 - GGG - loop2 - GGG - loop3 - GGG -3’. PCA of these data sets supported a series of correlations between the variables studied. First, the association of loop length with thermodynamic stability and quadruplex structure was corroborated. Secondly, it is proposed that the addition of ethylene glycol produces a stronger stabilization on those sequences showing long loop1 and/or loop3. Thirdly, it is proposed that a low content of adenine in loop1 and/or loop3 will produce an increase in the stability of G-quadruplex and its related Watson–Crick duplex.