Synthesis of a non-cationic, water-soluble perylenetetracarboxylic diimide and its interactions with G-quadruplex-forming DNA

A number of N,N'-disubstituted perylenetetracarboxylic diimides have been reported to bind effectively to DNA that adopts G-quadruplex motifs. In some cases, this binding may actively drive the transition from single-strand DNA to the quadruplex form. The perylenediimides in the reported cases all have amine-containing side chains, which are thought to interact with the grooves of the quadruplex and help dictate the selectivity of these compounds for quadruplex versus duplex DNA. We synthesized a polyethyleneglycol-swallowtailed (PEG-tailed) perylenediimide that is water-soluble even though it is uncharged. Binding to duplex and quadruplex DNA of this perylenediimide was studied by fluorescence quenching titrations under a variety of salt conditions, and the compound's effect on quadruplex formation was studied by non-denaturing gel electrophoresis. Our results indicate that while the molecule binds to single-stranded DNA quite effectively and with selectivity, it does not drive the transition of the DNA to the tetrameric quadruplex structure, supporting the idea that charge neutralization is a key component of perylene compounds that stabilize tetrameric quadruplexes.     

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR.

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.     

Human telomeric G-quadruplex DNA interactions of N-phenanthroline glycosylamine copper(II) complexes

We report in this article the interactions of five N-(1,10-phenanthrolin-5-yl)-β-glycopyranosylamine copper(II) complexes with G-quadruplex DNA. Specifically, the interactions of these compounds with a human telomeric oligonucleotide have been assessed by fluorescence-based assays (FRET melting and G4-FID), circular dichroism and competitive equilibrium dialysis experiments. The metal complexes bind and stabilize G-quadruplex DNA structures with apparent association constants in the order of 10⁴–10⁵ M⁻¹ and the affinity observed is dependent on the ionic conditions utilized and the specific nature of the carbohydrate moiety tethered to the 1,10-phenanthroline system. The compounds showed only a slight preference to bind G-quadruplex DNA over duplex DNA when the quadruplex DNA was folded in sodium ionic conditions. However, the binding affinity and selectivity, although modest, were notably increased when the G-quadruplex DNA was folded in the presence of potassium metal ions. Moreover, the study points towards a significant contribution of groove and/or loop binding in the recognition mode of quadruplex structures by these non-classical quadruplex ligands. The results reported herein highlight the potential and the versatility of carbohydrate bis-phenanthroline metal-complex conjugates to recognize G-quadruplex DNA structures.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.     

Photoisomerizable arylstilbazolium ligands recognize parallel and antiparallel structures of G-quadruplexes

The photoisomerization and DNA interaction studies of three arylstilbazolium derivatives with various samples of nucleic acids (duplexes, triplexes and tetraplexes) are reported. The equilibrium dialysis study revealed high binding affinities of ligands to tetraplex structures. The quadruplex-binding affinity could be switched by light, e.g., the E,E and E,Z isomers of 1,4-bis(vinylquinolinium)benzene (1) interacted with parallel and antiparallel tetraplexes exhibiting different binding selectivity. The E,Z-1 showed higher binding preference for c-myc DNA (a propeller-type quadruplex), whereas the E,E-1 favorably interacted with telomeric DNA (a basket-type quadruplex). The presence of quadruplex DNA hampered photoisomerization of quadruplex-bound ligand.     

Benzofuroquinoline Derivatives Had Remarkable Improvement of their Selectivity for Telomeric G-Quadruplex DNA over Duplex DNA upon Introduction of Peptidyl Group

In order to improve the selectivity of 5-N-methyl quindoline (cryptolepine) derivatives as telomeric quadruplex binding ligands versus duplex DNA, a series of peptidyl-benzofuroquinoline (P-BFQ) conjugates (2a-2n) were designed and synthesized. Their interactions with telomeric quadruplex and duplex DNA were examined by using the fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism spectroscopy (CD), and molecular modeling studies. Introduction of a peptidyl group at 11-position of the aromatic benzofuroquinoline scaffold not only effectively increased its binding affinity, but also significantly improved its selectivity toward telomeric quadruplex versus duplex DNA. Combined with the data for their inhibitory effects on telomerase activity, their structure-activity relationships (SARs) studies showed that the types of amino acid residues and the length of the peptidyl side chains were important for the improvement of their interactions with the telomeric G-quadruplex. Long-term exposure of human cancer cells to 2c showed a remarkable cessation in population growth and cellular senescence phenotype, and accompanied by a shortening of the telomere length.     

A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid fluorescent probes

Although quadruplex nucleic acids are thought to be involved in many biological processes, they are massively overwhelmed by duplex DNA in the cell. Small molecules, able to probe quadruplex nucleic acids with high optical selectivity, could possibly achieve the visualization of these processes. The aim of the method described herein is to evaluate quickly the optical selectivity of quadruplex nucleic acid probes, in isothermal conditions, using widely available materials, small quantities of oligonucleotides and virtually any kind and quantity of biological competitor. The assay relies on the use of streptavidin-coated paramagnetic particles and biotinylated quadruplex forming oligonucleotides, allowing a quick and easy separation of the quadruplex target from the competitor. In the present study, two quadruplex nucleic acids (the DNA and RNA human telomeric repeats) have been used as targets while a duplex DNA oligonucleotide, total DNA, total RNA, another quadruplex nucleic acid and a protein have been used as competitors. The optical selectivity of various probes, displaying different photophysical properties and binding selectivities, has been successfully examined, allowing the identification of a best candidate for further cell microscopy experiments. This assay allows a quick and reliable assessment of the labeling properties of a quadruplex binder in cellular environment conditions. It is an interesting alternative to gel electrophoresis experiments since it is performed in solution, has a well-resolved separation system and allows easy quantifications.     

Design and analysis of RNA structure-specific agents as potential antivirals

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants >10⁷M⁻¹), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.     

Differential binding of quadruplex structures of muscle-specific genes regulatory sequences by MyoD, MRF4 and myogenin

Four myogenic regulatory factors (MRFs); MyoD, Myf-5, MRF4 and Myogenin direct muscle tissue differentiation. Heterodimers of MRFs with E-proteins activate muscle-specific gene expression by binding to E-box motifs d(CANNTG) in their promoters or enhancers. We showed previously that in contrast to the favored binding of E-box by MyoD-E47 heterodimers, homodimeric MyoD associated preferentially with quadruplex structures of regulatory sequences of muscle-specific genes. To inquire whether other MRFs shared the DNA binding preferences of MyoD, the DNA affinities of hetero- and homo-dimeric MyoD, MRF4 and Myogenin were compared. Similarly to MyoD, heterodimers with E47 of MRF4 or Myogenin bound E-box more tightly than quadruplex DNA. However, unlike homodimeric MyoD or MRF4, Myogenin homodimers associated weakly and nonpreferentially with quadruplex DNA. By reciprocally switching basic regions between MyoD and Myogenin we demonstrated dominance of MyoD in determining the quadruplex DNA-binding affinity. Thus, Myogenin with an implanted MyoD basic region bound quadruplex DNA nearly as tightly as MyoD. However, a grafted Myogenin basic region did not diminish the high affinity of homodimeric MyoD for quadruplex DNA. We speculate that the dissimilar interaction of MyoD and Myogenin with tetrahelical domains in muscle gene promoters may differently regulate their myogenic activities.     

Insights into the RNA quadruplex binding specificity of DDX21

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2′OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2′OH of loop nucleotides.     

N-phenyl-carbazole-based two-photon fluorescent probes: strong sequence dependence of the duplex vs quadruplex selectivity

Herein we report on the synthesis and DNA recognition properties of a series of three N-phenyl carbazole-based light-up probes initially designed for two-photon absorption. The vinylic derivatives (Cbz-2Py, Cbz-3Py) display strong fluorescence enhancement when bound to various duplex- and quadruplex-forming oligonucleotides whereas the oxazole derivative is not fluorescent in DNA. Determination of affinity constants by fluorimetric titrations evidenced that Cbz-2Py has a clear preference for AT-rich duplex structures. Circular Dichroism (CD) measurements confirmed the sequence-dependent binding of this compound and suggest insertion in the minor groove as shown by a strong induced CD (ICD) signal and further supported by molecular modeling. Altogether the data indicate that duplex vs quadruplex selectivity of the dyes is strongly dependent on the sequence of the duplex. Finally, the dyes exhibit high two-photon absorption cross-sections (up to 540 GM in glycerol) and allow a fine and bright staining of nuclear DNA with low background fluorescence as shown by one and two-photon confocal microscopy imaging of fixed cells.     

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.     

In-silico modeling studies of G-quadruplex with soy isoflavones having anticancerous activity

Telomere forms t-loop and G-quadruplex as the protective structure and the formation of these structures hinder the telomerase enzyme action. The binding affinities of ligand which stabilize the G-quadruplex represent good correlation with telomerase inhibition depicted in the anti-cancerous action. Most of the potent G-quadruplex stabilizing compounds suffer from the poor drug like properties. Herein, natural dietary compounds isoflavones were taken for the theoretical study to examine their stabilizing effect on G-quadruplex structure. The experimental G-quadruplex complexes were reproduced to obtain and validate the theoretical parameters. The obtained theoretical binding energies are in significant correlation with the experimental data. Analysis of binding shows isoflavones to be groove binders, and differential nature of quadruplex grooves might be beneficial in the selectivity aspects. Among all, derrubone was found to have better selectivity as well as affinity for the G-quadruplex comparable to well known ligand TMPyP4. The GBSA rescoring result enlightens the various interaction terms involved in the binding process. Cumulative stabilizing effects coming from VDW, ES, and GB energy terms attest to optimal binding of derrubone molecule which can be considered as a lead for the higher phases of drug designing. These findings are of great value in terms of unexplored groove binding modes and the studied natural compounds might be helpful to direct the focus of synthetic chemists in designing of new generation of antitumor agents.     

Benzo(h)quinoline derivatives as G-quadruplex binding agents

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K_a in the range of 3 × 10⁵ M^?1 and with a 10-fold selectivity for quadruplex over duplex DNA.     

Double threading through DNA: NMR structural study of a bis-naphthalene macrocycle bound to a thymine-thymine mismatch

The macrocyclic bis-naphthalene macrocycle (2,7-BisNP), belonging to the cyclobisintercalator family of DNA ligands, recognizes T-T mismatch sites in duplex DNA with high affinity and selectivity, as evidenced by thermal denaturation experiments and NMR titrations. The binding of this macrocycle to an 11-mer DNA oligonucleotide containing a T-T mismatch was studied using NMR spectroscopy and NMR-restrained molecular modeling. The ligand forms a single type of complex with the DNA, in which one of the naphthalene rings of the ligand occupies the place of one of the mismatched thymines, which is flipped out of the duplex. The second naphthalene unit of the ligand intercalates at the A-T base pair flanking the mismatch site, leading to encapsulation of its thymine residue via double stacking. The polyammonium linking chains of the macrocycle are located in the minor and the major grooves of the oligonucleotide and participate in the stabilization of the complex by formation of hydrogen bonds with the encapsulated thymine base and the mismatched thymine remaining inside the helix. The study highlights the uniqueness of this cyclobisintercalation binding mode and its importance for recognition of DNA lesion sites by small molecules.     

Three HRDC domains differentially modulate Deinococcus radiodurans RecQ DNA helicase biochemical activity

RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNase D C-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNA-dependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.     

Aminoglycoside binding to Oxytricha nova telomeric DNA

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ～ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.     

Exploring the recognition of quadruplex DNA by an engineered Cys2-His2 zinc finger protein

We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.