Biophysical and biological properties of quadruplex oligodeoxyribonucleotides

Single-stranded guanosine-rich oligodeoxyribonucleotides (GROs) have a propensity to form quadruplex structures that are stabilized by G-quartets. In addition to intense speculation about the role of G-quartet formation in vivo, there is considerable interest in the therapeutic potential of quadruplex oligonucleotides as aptamers or non-antisense antiproliferative agents. We previously have described several GROs that inhibit proliferation and induce apoptosis in cancer cell lines. The activity of these GROs was related to their ability to bind to a specific cellular protein (GRO-binding protein, which has been tentatively identified as nucleolin). In this report, we describe the physical properties and biological activity of a group of 12 quadruplex oligonucleotides whose structures have been characterized previously. This group includes the thrombin-binding aptamer, an anti-HIV oligonucleotide, and several quadruplexes derived from telomere sequences. Thermal denaturation and circular dichroism (CD) spectropolarimetry were utilized to investigate the stability, reversibility and ion dependence of G-quartet formation. The ability of each oligonucleotide to inhibit the proliferation of cancer cells and to compete for binding to the GRO-binding protein was also examined. Our results confirm that G-quartet formation is essential for biological activity of GROs and show that, in some cases, quadruplex structures formed in the presence of potassium ions are significantly more active than those formed in the presence of sodium ions. However, not all quadruplex structures exhibit antiproliferative effects, and the most accurate factor in predicting biological activity was the ability to bind to the GRO-binding protein. Our data also indicate that the CD spectra of quadruplex oligonucleotides may be more complex than previously thought.     

Conformation and thermostability of oligonucleotide d(GGTTGGTGTGGTTGG) containing thiophosphoryl internucleotide bonds at different positions

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) (TBA) is an efficient tool for the inhibition of thrombin function. We have studied conformations and thermodynamic stability of a number of modified TBA oligonucleotides containing thiophosphoryl substitution at different internucleotide sites. Using circular dichroism such modifications were found not to disrupt the antiparallel intramolecular quadruplex specific for TBA. Nevertheless, the presence of a single thiophosphoryl bond between two G-quartet planes led to a significant decrease in the quadruplex thermostability. On the contrary, modifications in each of the loop regions either stabilized an aptamer structure or did not reduce its stability. According to the thrombin time test, the aptamer with thio-modifications in both TT loops (LL11) exhibits the same antithrombin efficiency as the original TBA. This aptamer shows better stability against DNA nuclease compared to that of TBA. We conclude that such thio-modification patterns are very promising for the design of anticoagulation agents.     

Synthesis and characterization of monomolecular DNA G-quadruplexes formed by tetra-end-linked oligonucleotides

Guanine-rich DNA sequences are widely dispersed in the eukaryotic genome and are abundant in regions with relevant biological significance. They can form quadruplex structures stabilized by guanine quartets. These structures differ for number and strand polarity, loop composition, and conformation. We report here the syntheses and the structural studies of a set of interconnected d(TG(4)T) fragments which are tethered, with different orientations, to a tetra-end-linker in an attempt to force the formation of specific four-stranded DNA quadruplex structures. Two synthetic strategies have been used to obtain oligodeoxyribonucleotide (ODN) strands linked with their 3'- or 5'-ends to each of the four arms of the linker. The first approach allowed the synthesis of tetra-end-linked ODN (TEL-ODN) containing the four ODN strands with a parallel orientation, while the latter synthetic pathway led to the synthesis of TEL-ODNs each containing antiparallel ODN pairs. The influence of the linker at 3'- or 5'-ODN, on the quadruplex typology and stability, in the presence of sodium or potassium ions, has been investigated by circular dichroism (CD), CD thermal denaturation, (1)H NMR experiments at variable temperature, and molecular modeling. All synthesized TEL-ODNs formed parallel G-quadruplex structures. Particularly, the TEL-ODN containing all parallel ODN tracts formed very stable parallel G-quadruplex complexes, whereas the TEL-ODNs containing antiparallel ODN pairs led to relatively less stable parallel G-quadruplexes. The molecular modeling data suggested that the above antiparallel TEL-ODNs can adopt parallel G-quadruplex structures thanks to a considerable folding of the tetra-end-linker around the whole quadruplex scaffold.     

Periodate oxidation in chemistry of nucleic acids: Dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides (Review)

The employment of periodate oxidation in the chemistry of nucleic acids and their components is reviewed. The reaction mechanism, structural requirements to substrates, and synthesis of dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides are discussed in the first part. The second part involves chemical, physico-chemical, and biological properties of the dialdehyde derivatives, as well as their use for the affinity modifications of proteins.     

Biological aspects of DNA/RNA quadruplexes.

Among the many unusual conformations of DNA and RNA, quadruplex structures, based on the guanine quartet, possess several unique properties. These properties, along with the general features of guanine quadruplexes, are described in the context of possible roles for these structures in biological systems. A variety of experimental observations supporting the notion that quadruplexes are important in vivo is presented, including proteins known to specifically bind to quadruplex structures, guanine-rich DNA, and RNA sequences endowed with the potential for forming quartet-based structures in telomeres and regulatory regions, such as gene promoters, quadruplexes as DNA aptamer folding motifs arising from in vitro selection experiments, and potential chemotherapeutic, quadruplex-forming oligonucleotides. Taken together, all of these observations argue cogently not only for the presence of quadruplexes in biological systems but also for their significance in terms of their roles in various biological processes.     

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.     

HIV-integrase aptamer folds into a parallel quadruplex: a thermodynamic study

Short guanine-rich sequences have a tendency to form quadruplexes that are stabilized by G-quartets with specific cation coordination. Quadruplexes are part of telomeres at the ends of chromosomes and play an important role in the regulation of gene expression. In addition, there is a strong interest in the therapeutic and biotechnological potential of quadruplex oligonucleotides. The HIV-integrase aptamer, d(GGGT)(4), demonstrated unusually favorable van't Hoff thermodynamics, and based on NMR studies the aptamer was proposed to fold into an antiparallel structure. Here we probed an apparent discrepancy between the NMR structure and the quadruplex topology suggested by circular dichroism (CD). Systematic thermodynamic analyses of d(GGGT)(4) and variants containing sequence modifications or missing specific nucleotides are consistent with a parallel quadruplex fold. CD studies carried out over a wide concentration range did not support a possible structural transition upon increasing strand concentration. Taken together, both optical and thermodynamic studies performed here strongly support a parallel fold for the d(GGGT)(4) aptamer.     

Unlocking G-quadruplex: Effect of unlocked nucleic acid on G-quadruplex stability

G-quadruplexes are common structural motifs in aptamers. UNA or unlocked nucleic acid is the latest nucleic acid modification. We have attempted to evaluate the impact of UNA modification on the structure and stability of G-quadruplex oligonucleotides for application in aptamer design. We show using CD spectroscopy that UNA modifications can cause structural transitions in some cases although they retain the inherent G- quadruplex signature. From UV melting studies we showed a position dependent effect of UNA modifications such that quadruplexes with UNA modified loops are further stabilized whereas UNA modifications in stem of the G-quadruplex significantly destabilize the structure. The impact of UNA modification on different nucleobases is also investigated. From the analysis of UV melting results, thermodynamic profile was computed and it was concluded that all the sequences are stable at 37 °C. Finally, a greater serum stability of the modified oligonucleotides in comparison with unmodified ones is also demonstrated. Overall, the position dependent effect of single UNA substitutions was observed and analysed.     

Antiproliferative activity of G-quartet-forming oligonucleotides with backbone and sugar modifications

Oligonucleotide-based therapies have considerable potential in cancer, viral, and cardiovascular disease therapies. However, it is becoming clear that the biological effects of oligonucleotides are not solely due to the intended sequence-specific interactions with nucleic acids. Oligonucleotides are also capable of interacting with numerous cellular proteins owing to their polyanionic character or specific secondary structure. We have examined the antiproliferative activity, protein binding, and G-quartet formation of a series of guanosine-rich oligonucleotides, which are analogues of GRO29A, a G-quartet forming, growth-inhibitory oligonucleotide, whose effects we have previously described [Bates P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377]. The GRO29A analogues include phosphorothioate (PS29A), 2'-O-methyl RNA (MR29A), and mixed DNA/2'-O-methyl RNA (MRdG29A) oligonucleotides. We demonstrate by UV spectroscopy that all of the modified analogues form stable structures, which are consistent with G-quartet formation. We find that the phosphorothioate and mixed DNA/2'-O-methyl analogues are able to significantly inhibit proliferation in a number of tumor cell lines, while the 2'-O-methyl RNA has no significant effects. Similar to the original oligonucleotide, GRO29A, the growth inhibitory oligonucleotides were able to compete with the human telomere sequence oligonucleotide for binding to a specific cellular protein. The less active MR29A does not compete significantly for this protein. On the basis of molecular modeling of the oligonucleotide structures, it is likely that the inactivity of MR29A is due to the differences in the groove structure of the quadruplex formed by this oligonucleotide. Interestingly, all GRO29A analogues, including an unmodified DNA phosphodiester oligonucleotide, are remarkably resistant to nuclease degradation in the presence of serum-containing medium, indicating that secondary structure plays an important role in biological stability. The remarkable stability and strong antiproliferative activity of these oligonucleotides confirm their potential as therapeutic agents.     

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.     

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.     

Effects of a 8-oxoadenosine incorporation on quadruplex structures: thermal stabilities and structural studies

The effects of incorporation of 8-oxoadenosine in two different truncations of human telomeric sequence forming quadruplex structures are reported. In order to characterise their structures, a combination of NMR and UV spectroscopy and computational techniques were used. Both oligonucleotides have been found to form fourfold symmetric quadruplex structures. As a tautomeric equilibrium between keto and enol forms of 8-oxoadenosine may establish in solution and intrinsic stabilities effects, such as internal H-bonds, for example, may determine the predominance of some particular tautomer, molecular modelling studies were performed on quadruplex structures containing both the tautomeric forms. Both molecules resulted to be thermally less stable than the natural.     

EFFECTS OF A 8-OXOADENOSINE INCORPORATION ON QUADRUPLEX STRUCTURES: THERMAL STABILITIES AND STRUCTURAL STUDIES

The effects of incorporation of 8-oxoadenosine in two different truncations of human telomeric sequence forming quadruplex structures are reported. In order to characterise their structures, a combination of NMR and UV spectroscopy and computational techniques were used. Both oligonucleotides have been found to form fourfold symmetric quadruplex structures. As a tautomeric equilibrium between keto and enol forms of 8-oxoadenosine may establish in solution and intrinsic stabilities effects, such as internal H-bonds, for example, may determine the predominance of some particular tautomer, molecular modelling studies were performed on quadruplex structures containing both the tautomeric forms. Both molecules resulted to be thermally less stable than the natural.     

Surface plasmon resonance for probing quadruplex folding and interactions with proteins and small molecules

Surface plasmon resonance is a technique for detecting binding events at the surface of a thin metal film. Through the commercial availability of instrumentation and sensor chips, the technique has found widespread application for determining the affinity and kinetics of macromolecular interactions. A variety of quadruplex forming oligonucleotides have been immobilized to sensor chips to permit analysis of their binding interactions with both small molecule and protein analytes. The fold of the quadruplex must be maintained through an appropriate choice of buffer, and care must be taken to ensure that data interpretation is not hampered by non-specific binding and adsorption of the analyte to the sensor surface and instrument. Affinity constants determined by surface plasmon resonance for interactions with quadruplexes correlate meaningfully with other methods, such as UV-visible and fluorescence titrations, enzyme linked immunosorbent assay, thermal melting studies and telomerase inhibition. Kinetic measurements of the association and dissociation of duplexes of quadruplex forming oligonucleotides and their complementary strands have enabled calculation of the folding and unfolding rates of the quadruplex itself, and determination of its stability as a function of buffer composition.     

Effects of 8-methylguanine on structure, stability and kinetics of formation of tetramolecular quadruplexes

Tetramolecular G-quadruplexes result from the association of four guanine-rich strands. Modification of the backbone strand or the guanine bases of the oligonucleotide may improve stability or introduce new functionalities. In this regard, the 8 position of a guanosine is particularly suitable for introduction of modifications since as it is positioned in the groove of the quadruplex structure. Modifications at this position should not interfere with structural assembly as would changes at Watson-Crick and Hoogsteen sites. In this study, we investigated the effect of an 8-methyl-2′-deoxyguanosine residue (M) on the structure and stability of tetramolecular parallel G-quadruplexes. In some cases, the presence of this residue resulted in the formation of unusual quadruplex structures containing all-syn tetrads. Furthermore, the modified nucleoside M at the 5′-end of the sequence accelerated quadruplex formation by 15-fold or more relative to the unmodified oligonucleotide, which makes this nucleobase an attractive replacement for guanine in the context of tetramolecular parallel quadruplexes.     

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.     

NMR structure of an antisense DNA.RNA hybrid duplex containing a 3'-CH(2)N(CH(3))-O-5' or an MMI backbone linker.

The solution structure of an antisense DNA.RNA hybrid duplex, d(CGCGTT-MMI-TTGCGC).r(GCGCAAAACGCG) (designated R4), containing an MMI backbone linker [3'-CH(2)N(CH(3))-O5'], is elucidated. The structural details of the MMI linker, its structural effects on the neighboring residues, and the molecular basis of the MMI effects are examined. The lipophilic N-methyl group of MMI is peripheral to the helix, assuming a conformation that is most stable with regard to the N-O torsion angle. The MMI linker promotes a 3'-endo conformation for the sugar moieties at both 3'- and 5'-adjacent positions and a backbone kink involving distant residues along the 3'-direction. Comparison of R4 with other analogous hybrid duplexes previously studied in this laboratory reveals a new family of low-energy helical conformations that can be accommodated in stable duplexes and a common feature of C3'-modified sugars for adopting a C3'-endo pucker. The results of these studies emphasize the interplay of several factors that govern the formation of stable hybrid duplexes and provide a basis for the understanding of the biological role of the MMI modifications, which are important building blocks for a family of promising chimeric antisense oligonucleotides.     

A Simple and Efficient Procedure for the Synthesis of an Alendronate- Oligonucleotide Conjugate via a Carbamate Linker

Abstract

In order to combine the biological properties of oligonucleotides, a synthetic chemical modelized reaction was performed and the procedure then applied to the preparation of an alendronate-deoxyoligonucleotide conjugate through a carbamate linker.