The effect on quadruplex stability of North-nucleoside derivatives in the loops of the thrombin-binding aptamer

doi:10.1016/j.bmc.2012.06.005

Bioorganic & Medicinal Chemistry

Volume 20, Issue 14, 15 July 2012, Pages 4186-4193

https://doi.org/10.1016/j.bmc.2012.06.005 Get rights and content

Abstract

Modified thrombin-binding aptamers (TBAs) carrying uridine (U), 2′-deoxy-2′-fluorouridine (FU) and North-methanocarbathymidine (NT) residues in the loop regions were synthesized and analyzed by UV thermal denaturation experiments and CD spectroscopy. The replacement of thymidines in the TGT loop by U and FU results in an increased stability of the antiparallel quadruplex structure described for the TBA while the presence of NT residues in the same positions destabilizes the antiparallel structure. The substitution of the thymidines in the TT loops for U, FU and NT induce a destabilization of the antiparallel quadruplex, indicating the crucial role of these positions. NMR studies on TBAs modified with uridines at the TGT loop also confirm the presence of the antiparallel quadruplex structure. Nevertheless, replacement of two Ts in the TT loops by uridine gives a more complex scenario in which the antiparallel quadruplex structure is present along with other partially unfolded species or aggregates.

Graphical abstract

Introduction

Nucleic acid sequences rich in guanines are predisposed to form higher order structures because of the capacity of guanine bases to self associate via Hoogsteen hydrogen bonds to form planar tetrads. Several tetrads placed next to another stack in a very effective G-quadruplex structure.1, 2 G-rich DNA or RNA sequences have been identified in biologically relevant regions of the genome.³ These sequences are present in telomeres⁴ and in promoter regions of oncogenes such as c-myc,⁵ c-Kit,⁶ K-ras⁷ and others.

Quadruplex structures differ in the number of associated strands and their orientation, and in the topology and conformation of the loops that connect the G-tetrads. When the guanine strands are oriented in opposite directions with loops above and below the terminal G-tetrad, the quadruplex is ‘antiparallel’. In this type of quadruplex, half of the guanines are oriented in syn around the glycosyl bond and the other half in anti.8, 9 In contrast, when all the guanines are oriented in the same direction with loops located on the side of the G-tetrads, the quadruplex is ‘parallel’. In this case, the glycosyl bonds of nucleosides adopt an anti-orientation.2, 8 Mixed parallel/antiparallel quadruplexes have also been observed.¹⁰ Moreover intramolecular quadruplexes can exhibit several G-quadruplex conformations. For example, a number of studies have demonstrated that human telomere regions forms various G-quadruplex structures.11, 12, 13, 14

While many of the studies have addressed modifications in the guanines of the G-tetrad and their implication in folding topology and molecularity,15, 16 less attention has been paid to the conformation of the nucleosides at the loop regions.

Loops play a key role in determining the nature of the folding and stability of the G-quadruplex.17, 18, 19 Both loop length and sequence are crucial in the folding and can either stabilize or destabilize the quadruplex structure.¹⁷

Several authors have studied nucleotide substitution in quadruplex forming oligonucleotides containing a single base loop²⁰ or different loop length compositions.18, 21, 22 In addition studies involving quadruplexes with non-nucleosidic linkers instead of loops revealed the formation of parallel quadruplexes.²³ Another important point is the loop–loop interactions such as hydrogen bonding and stacking, which directly affect quadruplex topology.24, 25 In addition to these factors, cations also make a key contribution to the folding and stability of the quadruplex.26, 27

Although DNA G-quadruplex structure has been studied intensely, little is known about the stability and folding of RNA quadruplex forming oligonucleotides. These RNA quadruplexes are characterized by a parallel orientation of strands, which results from the preferential anti-orientation of their glycosyl bonds.²⁸

Several authors studied quadruplex formation by DNA/RNA hybrid analogues of G₄T₄G₄, in particular the role of the dT₄ loop and the effect of its substitution for rU₄.²⁹ Moreover, the substitution of a T for U in the tetramolecular TG₄T quadruplex, was studied demonstrating that it has a parallel orientation of the four strands.³⁰ This substitution favored the formation of dimeric structures d[(UG₄T)₄]₂ in the presence of K⁺ and NH₄⁺. In addition, the stabilization of 2′-deoxy-2′-fluoro-d-arabinonucleic acids (2′-F-ANA) on various positions including the loops of G-quadruplex structures has been studied.³¹

Normally, the ring conformation or puckering can be conveniently described by two parameters, namely the pseudorotation angle and the amplitude of the puckering. In nucleosides and nucleotides structures, as well as in oligonucleotides, the sugar pseudorotation angle is populated essentially by two types of conformation, referred to as the North and South.32, 33 In the B-form of DNA, 2′-deoxyribose sugars exhibit a preferential 2′-endo/3′-exo (South) conformation, while A-form helices and RNA tend to have 2′-exo/3′-endo (North) puckering.

Thrombin-binding aptamer (TBA) is a 15-mer DNA structure d(GGTTGGTGTGGTTGG) that specially binds to human thrombin.34, 35 NMR spectroscopy reveals that this structure adopts a uniquely folded structure with two stacked G-tetrads connected through edge-loops (one TGT and two TT loops) involving antiparallel alignment of the adjacent strands.36, 37, 38 The TBA loops are crucial for the thrombin-binding and recognition. NMR shows that the two TT loops interact with the fibrinogen recognition site (exosite I) of one thrombin molecule and the TGT loop interacts with the heparin-binding site (exosite II) of a second thrombin molecule.³⁵

TBA is characterized by nucleotides with a South (S) sugar pucker. The loops of the aptamer adopt the anti orientation while the guanines on the same G-tetrad plane display alternating syn/anti conformations with respect to the glycosyl torsion angle.³⁶ The introduction of a nucleotide with opposite glycosyl orientation directly affects the stability of TBA.15, 16 Several authors have also studied the influence of the loop length and composition of the loops on the folding of the aptamer.³⁹ In addition, cation-binding to TBA is important for driving TBA stability⁴⁰ and recently it has been described that a multiple pathway process is affected by the type of cation.²⁷ According to NMR experiments, TBA denaturation begins with the opening of the G–G base pairs that are not protected by a loop, followed by the opening the TGT loop.⁴¹

Here we studied the conformational changes produced in TBA when the thymidines located in the loops are substituted by ribonucleosides (uridine and 2′-deoxy-2′-fluorouridine) or by the conformationally-restricted pseudonucleoside North-methanocarbathymidine.⁴²

2′-Fluoro-2′-deoxy ribonucleosides show a strong preference >90% for the North type conformer in solution.43, 44, 45 North-methanocarbathymidine is a conformationally-restricted pseudonucleoside with a carbocyclic bicyclo[3.1.0]hexane ring. North or South-locked platforms can be prepared by shifting the position of the cyclopropane ring.42, 46, 47

We found that the North conformation of these derivatives directly interferes with TBA folding and stability, thereby confirming the relevance of loop flexibility in TBA folding.

Section snippets

Synthesis of modified thrombin-binding aptamer oligonucleotides

Thymidines present in the loop regions of TBA were replaced by several uridine (U), 2′-deoxy-2′-fluorouridine (FU) or North-methanocarbathymidine (NT) residues (Fig. 1A). To this end, several oligonucleotide sequences were prepared by solid-phase phosphoramidite synthesis. U, FU and NT phosphoramidites were used for the synthesis of modified TBA sequences. The 2′-hydroxyl group of uridine was protected with the t-butyldimethylsilyl (TBDMS) group.

First, the two thymidines located in the central

Conclusions

In summary, here we have studied the substitution of several thymidines located at the loop positions for derivatives with a North conformation (uridine, 2′-deoxy-2′-fluorouridine and North-methanocarbathymidine). Our findings demonstrate that changes in the conformation of the sugar puckering of the nucleotides present in loop regions are important for determining the folding and stability of TBA.

The central TGT loop of TBA is more tolerant to modifications (except for NT) than the two lateral

Materials

All the standard phosphoroamidites and reagents for DNA synthesis were purchased from Applied Biosystems and from Link Technologies. The remaining chemicals and solvents were from commercial sources (Sigma, Aldrich, Fluka) and were used without further purification.

Oligonucleotide synthesis and purification

The assembly of the modified TBAs was performed on a DNA/RNA synthesizer by solid-phase 2-cyanoethylphosphoroamidite chemistry. Commercially available controlled pore glass functionalized with 5′-O-DMT-2′-deoxyguanosine was used as

Acknowledgments

This work was partially supported by Grants from the Spanish Ministerio de Ciencia e Innovación MICINN (CTQ2010-20541), the Generalitat de Catalunya, (2009/SGR/208), the University of Milano (PUR 2009 Funds) and PRIN09 (2009Prot.2009J54YAP_005). RF is a recipient of a FPI predoctoral contract (MICINN). Collaborative research was funded by a Cost action (G4net, MP0802) and an Italian-Spanish collaborative action (IT2009-0067). CIBER-BBN is an initiative funded by the VI National R&D&i Plan

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Citation Excerpt :
Notably, lower Tm values were found for both substitutions at position 15, with higher effects using S(syn)dG (Table 3), suggesting that the N-glycosyl conformation is more crucial for TBA stability than the sugar puckering (Saneyoshi et al., 2009). In turn, Aviñó et al. evaluated the insertion of N-methanocarba nucleosides in the loop regions at positions 7 and 9 or 4 and 13 (TBA-NT7,9 and TBA-NT4,13, respectively), providing in both cases TBA derivatives with a dramatic reduction of the G4 thermal stability compared to their unmodified counterpart (Table 3) (Aviñó, Mazzini, et al., 2012). Peng and coworkers evaluated the insertion of 2′-deoxy-2′-fluoro-D-arabinonucleosides (2′F-araN or 2′F-ANA, Fig. 5e) – able to confer DNA-like (South/East) sugar conformations along with increased nuclease resistance – both in the G-quartets and the loop regions of the TBA sequence (Kalota et al., 2006; Trempe et al., 2001).
First studies on thrombin-inhibiting DNA aptamers were reported in 1992, and since then a large number of anticoagulant aptamers has been discovered. TBA – also named HD1, a 15-mer G-quadruplex (G4)-forming oligonucleotide – is the best characterized thrombin binding aptamer, able to specifically recognize the protein exosite I, thus inhibiting the conversion of soluble fibrinogen into insoluble fibrin strands. Unmodified nucleic acid-based aptamers, in general, and TBA in particular, exhibit limited pharmacokinetic properties and are rapidly degraded in vivo by nucleases. In order to improve the biological performance of aptamers, a widely investigated strategy is the introduction of chemical modifications in their backbone at the level of the nucleobases, sugar moieties or phosphodiester linkages. Besides TBA, also other thrombin binding aptamers, able to adopt a well-defined G4 structure, e.g. mixed duplex/quadruplex sequences, as well as homo- and hetero-bivalent constructs, have been identified and optimized.
Considering the growing need of new efficient anticoagulant agents associated with the strong therapeutic potential of these thrombin inhibitors, the research on thrombin binding aptamers is still a very hot and intriguing field. Herein, we comprehensively described the state-of-the-art knowledge on the DNA-based aptamers targeting thrombin, especially focusing on the optimized analogues obtained by chemically modifying the oligonucleotide backbone, and their biological performances in therapeutic applications.
TBA loop mapping with 3′-inverted-deoxythymidine for fine-tuning of the binding affinity for α-thrombin
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TBA is a 15-mer DNA aptamer for human α-thrombin, and its three T-rich loops are involved in the binding interactions with thrombin differently. In order to clarify their specific spatial locations in the binding interactions and search for more favourable positions, here a systematic investigation of all the loop residues was conducted with 3′-inverted thymidine (ⁱT), by which both unnatural 3′–3′- and 5′–5′-linkages for each incorporation were introduced in the tertiary structure. The changes in T_m values and CD spectra revealed that motifs T3T12 and T4T13 are structurally distinct. Longer anti-clotting time was obtained for the T3 and T12 modifications, respectively, while T4 and T13 were completely intolerant with such changes, in terms of stability and binding to thrombin. In particular, the increased affinity bindings and longer anti-clotting time were obtained with the replacement on the central loop T7G8T9, which were closely related to the existence of a monovalent ion, K⁺ or Na⁺, consistently with the supposed binding site of these ions in TBA. It is worthwhile to note that both the subtle variations of the loop residues induced by ⁱT and the monovalent ions determined the interacting residues of TBA and the binding strength rather than the thermal stability of the TBA structure.
Favorable 2′-substitution in the loop region of a thrombin-binding DNA aptamer
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Simple 2′-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (T → 2′-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting.
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Unlike RNA-TBA (rTBA), iso-rTBA, the 2′-5′-linked RNA-thrombin-binding aptamer, is a functional equivalent of TBA
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The effect on quadruplex stability of North-nucleoside derivatives in the loops of the thrombin-binding aptamer

Abstract

Graphical abstract

Introduction

Section snippets

Synthesis of modified thrombin-binding aptamer oligonucleotides

Conclusions

Materials

Oligonucleotide synthesis and purification

Acknowledgments

Curr. Opin. Struct. Biol.

FEBS Lett.

Biochimie

Bioorg. Med. Chem.

J. Biol. Chem.

J. Mol. Biol.

Bioorg. Med. Chem.

Methods Enzymol.

Nucleic Acids Res.

Quadruplex Nucleic Acids

Biopolymers

BioEssays

Nucleic Acids Res.

Biochemistry

Nucleic Acids Res.

Biol. Chem.

J. Am. Chem. Soc.

J. Am. Chem. Soc.

Nucleic Acids Res.

Nucleic Acids Res.

Proc. Natl. Acad. Sci. U.S.A.

Nucleic Acids Res.

J. Am. Chem. Soc.

Biochemistry

J. Am. Chem. Soc.