G-quadruplex and i-motif are mutually exclusive in ILPR double-stranded DNA

G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin.     

Sequence-specific recognition of double-stranded DNA by synthetic minor groove binder conjugates. toward the construction of artificial site-specific deoxyribonucleases

Bis-conjugates of hairpin N-methylpyrrole/N-methylimidazole oligocarboxamide minor groove binders (MGB) possessing enhanced affinity and sequence-specificity for dsDNA were synthesized. Two hairpin MGBs were connected by their N-termini via an aminodiacetate linker. The binding of bis-MGB conjugates to the target DNA was studied by gel mobility retardation, footprinting, and circular dichroism; their affinity and binding mode in the DNA minor groove were determined. In order to functionalize the bis-MGB conjugates, DNA-cleaving agents such as phenanthroline or bipyridine were attached. Effective site-specific cleavage of target DNA in the presence of Cu(2+) ions was observed.     

Thermodynamics of interaction of phthalocyanine-oligonucleotide conjugates with single- and double-stranded DNA

Thermodynamics of interaction of phthalocyanine-oligonucleotide conjugates with single- and double-stranded DNA resulting in formation of duplexes and triplexes was measured by UV melting method. It was shown that a phthalocyanine moiety of conjugates stabilized the formation of duplexes and triplexes.     

Perylene ligand wrapping G-quadruplex DNA for label-free fluorescence potassium recognition

A perylene ligand, N,N-bis-(1-aminopropyl-3-propylimidazol salt)-3,4,9,10-perylene tetracarboxylic acid diimide ligand (PDI), which consisted of π-conjugated perylene moiety and hydrophilic side chains with positively charged imidazole rings, was used to wrap G-quadruplex for fluorescence turn-on K(+) recognition. Electrostatic attraction between PDI's positively charged imidazole rings and DNA's negatively charged phosphate backbones enabled PDI to accumulate on DNA. Upon trapping K(+), these G-rich DNA sequences transitioned to G-quadruplex. Subsequently, PDI ligands wrapped G-quadruplex, in which the flat aromatic core of PDI ligand interacted with G-quartet through π-π stacking and the side chains were positioned in grooves through electrostatic interactions. Consequently, the interaction mode change and conformational transition from PDI stacked G-sequence to PDI wrapped G-quadruplex led to PDI fluorescence enhancement, which was readily monitored as the detection signal. This strategy excluded the sequence tagging step and exhibited high selectivity and sensitivity towards K(+) ion with the linear detection range of 10-150nM. Besides, PDI ligands may hold diagnostic and therapeutic application potentials to human telomere and cancer cells.     

Selective recognition of a DNA G-quadruplex by an engineered antibody

Particular guanine rich nucleic acid sequences can fold into stable secondary structures called G-quadruplexes. These structures have been identified in various regions of the genome that include the telomeres, gene promoters and UTR regions, raising the possibility that they may be associated with biological function(s). Computational analysis has predicted that intramolecular G-quadruplex forming sequences are prevalent in the human genome, thus raising the desire to differentially recognize genomic G-quadruplexes. We have employed antibody phage display and competitive selection techniques to generate a single-chain antibody that shows >1000-fold discrimination between G-quadruplex and duplex DNA, and furthermore >100-fold discrimination between two related intramolecular parallel DNA G-quadruplexes. The amino acid sequence composition at the antigen binding site shows conservation within the light and heavy chains of the selected scFvs, suggesting sequence requirements for G-quadruplex recognition. Circular dichroism (CD) spectroscopic data showed that the scFv binds to the prefolded G-quadruplex and does not induce G-quadruplex structure formation. This study demonstrates the strongest discrimination that we are aware of between two intramolecular genomic G-quadruplexes.     

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

Background

Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.

Methods

Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.

Results

These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.

Conclusions

These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.

General observation

This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.     

Tuning the selectivity of N-alkylated styrylquinolinium dyes for sensing of G-quadruplex DNA

Selective and sensitive detection of G-quadruplex DNA structures is an important issue and attracts extensive interest. To this end, numerous small molecular fluorescent probes have been designed. Here, we present a series of N-alkylated styrylquinolinium dyes named Ls-1, Ls-2 and Ls-3 with varying side groups at the chain end. We found that these dyes exhibited different binding behaviors to DNAs, and Ls-2 with a sulfonato group at the chain end displayed sensitivity and selectivity to G-quadruplex DNA structures in vitro. The characteristics of this dye and its interaction with G-quadruplex DNA were comprehensively investigated by means of UV–vis spectrophotometry, fluorescence, circular dichroism and molecular docking. Furthermore, confocal fluorescence images and MTT assays indicated dye Ls-2 could pass through membrane and enter the living HepG2 cells with low cytotoxicity.     

Direct site-specific cleavage of double-stranded DNA by conjugates of bleomycin A5 with triplex-forming oligonucleotide

Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5′-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3′-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blmp15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (>90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds.     

Hybridization kinetics between immobilized double-stranded DNA probes and targets containing embedded recognition segments

We have investigated the time-dependent strand displacement activity of several targets with double-stranded DNA probes (dsProbes) of varying affinity. Here, the relative affinity of various dsProbes is altered through choices in hybridization length (11-15 bases) and the selective inclusion of center mismatches in the duplexes. While the dsProbes are immobilized on microspheres, the soluble, 15 base-long complementary sequence is presented either alone as a short target strand or as a recognition segment embedded within a longer target strand. Compared to the short target, strand displacement activity of the longer targets is slower, but still successful. Additionally, the longer targets exhibit modest differences in the observed displacement rates, depending on the location of recognition segment within the long target. Overall, our study demonstrates that the kinetics of strand displacement activity can be tuned through dsProbe sequence design parameters and is only modestly affected by the location of the complementary segment within a longer target strand.     

Detection of G-quadruplex DNA in mammalian cells

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.     

G-quadruplex DNA structures--variations on a theme

To be functional, nucleic acids need to adopt particular three-dimensional structures. For a long time DNA was regarded as a rigid and passive molecule with the sole purpose to store genetic information, but experimental data has now accumulated that indicates the full dynamic repertoire of this macromolecule. During the last decade, four-stranded DNA structures known as G-quadruplexes, or DNA tetraplexes, have emerged as a three-dimensional structure of special interest. Motifs for the formation of G-quadruplex DNA structures are widely dispersed in eukaryotic genomes, and are abundant in regions of biological significance, for example, at telomeres, in the promoters of many important genes, and at recombination hotspots, to name but a few in man. Here I explore the plethora of G-quadruplex DNA structures, and discuss their possible biological functions as well as the proteins that interact with them.     

G-quadruplex DNA aptamers generated for systemin

Ligands specific to bioactive molecules play important roles in biomedical researches and applications, such as biological assay, diagnosis and therapy. Systemin is a peptide hormone firstly identified in plant. In this paper we report the selection of a group of DNA aptamers that can specifically bind to systemin. Through comparing the predicted secondary structures of all the aptamers, a hairpin structure with G-rich loop was determined to be the binding motif of these aptamers. The G-rich loop region of this binding motif was further characterized to fold into an antiparallel G-quadruplex by truncation-mutation assay and CD spectrum. The apparent equilibrium dissociation constant (K(d)) of one strong binding sequence (S-5-1) was measured to be 0.5 μM. The specificity assay shows that S-5-1 strongly bind to whole systemin, weakly bind to truncated or mutated systemin and does not bind to the scrambled peptide with the same amino acid composition as systemin. The high affinity and specificity make S-5-1 hold potentials to serve as a molecular ligand applied in detection, separation and functional investigation of systemin in plants.     

DNA sequence recognition by Hoechst 33258 conjugates of hairpin pyrrole/imidazole polyamides

A series of hairpin pyrrole/imidazole polyamides linked to a Hoechst 33258 (Ht) analogue (5-7) were synthesized on solid-phase by adopting an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. The dsDNA binding properties of Ht-polyamides 5-7 were determined by thermal denaturation experiments. Hairpin Ht-polyamides 5-7 bound to dsDNA sequences 16 and 18 show DeltaTm values that are 14-18 degrees higher than linear Ht-polyamides bound to the same sequences. All three Ht-polyamides were found to be selective for their 9-bp match dsDNA sequences, supporting a relative stronger interaction of an Im/Py anti-parallel dimer with an appropriately positioned G/Cbp rather than sequences containing only A/Tbps. In addition, Ht-polyamides 5 and 7 showed a 20-fold preference for a properly placed G/Cbp over a C/Gbp, while 6 showed a 10-fold preference.     

Requirement of beta-alanine components in sequence-specific DNA alkylation by pyrrole-imidazole conjugates with seven-base pair recognition

To investigate the effect of incorporation of beta-alanine in alkylating N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide, seco-CBI conjugates 2-8 were synthesized by an Fmoc solid-phase method and subsequent coupling with an alkylating moiety. DNA-alkylating activities of conjugates 2-8 were evaluated by high-resolution denaturing gel electrophoresis with 202-base pair (bp) DNA fragments. Alkylation by conjugates 2 and 3, which have antiparallel pairings of beta-alanine (beta) opposite beta (beta/beta) and Py/beta, occurred mainly at the adenine (A) of the matching sequences, 5'-AGCTCCA-3' (site 1) and 5'-AGCACCA-3' (site 3). However, conjugate 4, with beta/Py, did not show any DNA-alkylating activities. Similarly, conjugate 5, which possessed a Py/Py pair, weakly alkylated the matching sites at micromolar concentrations. Conjugates 6 and 7, which possessed beta/beta and Py/beta pairs, respectively, alkylated at the A of the matching sequences, 5'-ACTACCA-3' (site 2) and 5'-ACAACCA-3' (site 4). In contrast, conjugated 8, with a Py/Py pair, showed lower activity and less alkylated DNA at sites 2 and 4 with mismatched alkylation at site 1 at a higher concentration than that of 6 and 7. These results demonstrate that incorporation of beta-alanine is required for the sequence-specific alkylation by seco-CBI Py-Im conjugates with a seven-base pair sequence.