Structural transformation induced by locked nucleic acid or 2′–O-methyl nucleic acid site-specific modifications on thrombin binding aptamer
© Liu and Li; licensee Chemistry Central Ltd. 2014
Received: 2 October 2013
Accepted: 13 March 2014
Published: 19 March 2014
Locked nucleic acid (LNA) and 2'–O-methyl nucleic acid (OMeNA) are two of the most extensively studied nucleotide derivatives in the last decades. However, how they affect DNA quadruplex structures remains largely unknown. To explore their possible biological affinities for quadruplexes, we investigated how LNA- or OMeNA-substitutions affect G-quadruplex structure formation using a thrombin binding aptamer (TBA), the most studied extracorporal G-quadruplex-forming DNA sequence, which is frequently modified to increase its analytical performance.
The experimental results showed that when two or more nucleotides were substituted with LNA or OMeNA, the anti-parallel TBA structure was transformed into an unstructured random conformation in a 50 mM K+ environment; OMeNA appeared to have greater power to induce this transformation. However, the native TBA was unstructured in a 50 mM Ca2+ environment, whereas four or more LNA- or OMeNA- substitutions could convert this unstructured TBA into a parallel quadruplex structure. PAGE mobility measurements suggested that these TBAs might be a dimeric form.
LNA or 2'-OMeNA site-specific modifications induced G-quadruplex structural transformation of TBA, which enriched our understanding of the intrinsic G-quadrupex forming property and affinity of LNA and OMeNA modifications. This study demonstrates possible applications in the regulation of gene expression (i.e. manual intervention of gene therapy), genetic analyses, molecular diagnosis and the construction of nano-scale biostructures.
KeywordsG-quadruplex TBA LNA 2'-OMeNA Conformation
Primer oligonucleotide sequences of selected aptamers
g2 (2, 11)
g2 (5, 11)
g2 (10, 11)
g2 (11, 14)
g4 (2, 5, 11, 14)
g8 (1, 2, 5, 6, 10, 11, 14, 15)
LNA- or OMeNA-modifications induced TBA structural transition from an antiparallel G-quadruplex to a random strand in a K+environment
LNA- or OMeNA-modifications tend to form a parallel G-quadruplex topology of TBA in a Ca2+environment
Oligonucleotide modifications have attracted great attention in structural and functional research, in which LNA and OMeNA are quite widely used in aptamer modifications. Previous studies have shown that single LNA-substitutions for G2, T4, G5, T7, G8 or G15 have no effect on TBA structural alterations [19, 20], and that LNA-substituted TBAs on two G-tetrad planes could have different structures coexisting in solution . These results might be correlated with glycosidic bond angles determined by the positions where LNA nucleotides were incorporated, as it is the preference of LNA nucleotides to adopt an anti-glycosidic conformation based on the C3′-endo sugar pucker . OMeNA-substituted TBAs, however, have been rarely studied before. In this study, we report for the first time that structural transitions of TBA are regulated by multisite-specific LNA or 2′-OMeNA substitutions. The results showed that the native structure of TBA changed from an anti-parallel G-quadruplex to an unstructured form in a K+ environment, and from an unstructured form to a parallel G-quadruplex in a Ca2+ environment. The molecular mechanism may involve the syn- or anti-conformation of guanine residues of G-quartet construction (Additional file 2: Figure S1), and optional modified sites (except for ion effect). Also, Additional file 1: Figure S2 shows the assumed possible conformations of multi-LNA- or OMeNA-modified TBAs. These results suggest that the G-quadruplex conformation mostly depends on the glycosidic conformation of guanine residues in the G-tracts, which corresponds with a previous report . Otherwise, different metal ions may also play an unneglected role in G-quadruplex structure formation . Accordingly, specific regulatory mechanisms of G-quadruplex conformation may exist, by which the current experimental evidence provides us an opportunity to create polymorphic G-quadruplexes using nucleotide derivative substitutions at selected positions in conjunction with different ionic conditions.
In summary, we monitored diverse quadruplex structural transformations through LNA or OMeNA site-specific modifications on TBA, which were regulated by the syn- or anti-conformation of guanine residues of G-quartet construction and optional modified sites. Moreover, both LNA and OMeNA substitutions could alter the TBA G-quadruplex structure, and OMeNA might have more destructive power than LNA to convert TBA into a structureless form in a K+ environment; in a Ca2+ environment, LNA- or OMeNA substitutions could transform the native unstructured TBA into a parallel G-quadruplex conformation. These observations enrich our understanding of the nature of LNA and OMeNA substitutions and the behavior of G-quadruplex structure formation. The significance of these structural transitions may benefit the regulation of gene expression (i.e. manual intervention of gene therapy), genetic analyses, molecular diagnosis and the construction of nano-scale biostructures.
Preparation of oligonucleotides
The oligonucleotides were purchased from TaKaRa Biotech Ltd. (Dalian China). The lyophilized oligonucleotides were dissolved in 1 × TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 7.2) to give a stock solution concentration of 100 μM. Before starting the experiments, all of the oligonucleotide samples were denatured (5 min at 95°C) to remove aggregates. The sample was then left to cool to room temperature. The formation of quadruplexes was followed at 4°C, unless stated otherwise.
Circular dichroism spectroscopy
Circular dichroism measurements were carried out on a Jasco J-810 spectropolarimeter (Jasco, Easton, MD) in a quartz sample cell with an optical path length of 1 mm. 500 μL of sample solution was added and the cell was placed in a thermostable holder maintained at room temperature during the measurements. The CD spectra were representative of three averaged scans at a speed of 500 nm per minute, made from 340 nm to 220 nm. The final DNA concentration of 5 μM for the CD spectroscopic study was prepared in 1 × TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 7.2) with either 50 mM KCl or CaCl2, respectively. To ensure that all TBA aptamers adopted the same structure at the beginning of each experiment, a slow melting-cooling cycle at 0.2°C per minute was performed prior to each experiment. The unsubstituted TBA and modified-TBAs with identical sequences were assumed to have identical extinction coefficients.
Non-denatured PAGE electrophoresis
Different substituted TBA species were incubated with 1 × TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 7.2) in the presence of 50 mM KCl or CaCl2 at 37°C for 1 h, respectively. The 15% non-denatured PAGE gels were prepared by mixing 10 mL 30% acrylamide solution, 4 mL 5 × TB buffer (containing 250 mM KCl or CaCl2), 5.86 mL water, 140 μL 10% APS, and 13 μL TEMED. The 8 × 10 × 0.1 cm gel was polymerized within 45 min. A final concentration of 20 μM TBA solution (containing 2 μL stock solution, 2 μL loading buffer, 0.5 μL 1 M KCl or CaCl2, and 5.5 μL water) was loaded onto gels. Gels were run in 1 × TB buffer (containing 50 mM KCl or 50 mM CaCl2) for 1.5 h at 100 V at 4°C. The gels were then stained with Stains-All (Sigma, USA), washed three times, and images were recorded using a Personal Scanner (Model Z320, FangZheng, China).
Locked nucleic acid
2'–O-methyl nucleic acid
Thrombin binding aptamer
Ethylene Diamine Tetraacetic Acid
Polyacrylamide gel electrophoresis.
This work was supported by the Natural Science Foundation of China (No. 31070705).
- Wilson C, Keefe AD: Building oligonucleotide therapeutics using non-natural chemistries. Curr Opin Chem Biol. 2006, 10: 607-614. 10.1016/j.cbpa.2006.10.001.View ArticleGoogle Scholar
- Kaur H, Babu BR, Maiti S: Perspectives on chemistry and therapeutic applications of Locked Nucleic Acid (LNA). Chem Rev. 2007, 107: 4672-4697. 10.1021/cr050266u.View ArticleGoogle Scholar
- Prakash TP, Bhat B: 2′-Modified oligonucleotides for antisense therapeutics. Curr Top Med Chem. 2007, 7: 641-649. 10.2174/156802607780487713.View ArticleGoogle Scholar
- Yan Y, Yan J, Piao X, Zhang T, Guan Y: Effect of LNA- and OMeN-modified oligonucleotide probes on the stability and discrimination of mismatched base pairs of duplexes. J Biosci. 2012, 37: 233-241. 10.1007/s12038-012-9196-4.View ArticleGoogle Scholar
- Huppert JL: Hunting G-quadruplexes. Biochimie. 2008, 90: 1140-1148. 10.1016/j.biochi.2008.01.014.View ArticleGoogle Scholar
- Keniry MA: Quadruplex structures in nucleic acids. Biopolymers. 2000, 56: 123-146. 10.1002/1097-0282(2000/2001)56:3<123::AID-BIP10010>3.0.CO;2-3.View ArticleGoogle Scholar
- Qin Y, Hurley LH: Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie. 2008, 90: 1149-1171. 10.1016/j.biochi.2008.02.020.View ArticleGoogle Scholar
- Gomez-Marquez J: DNA G-quadruplex: structure, function and human disease. FEBS J. 2010, 277: 3451-10.1111/j.1742-4658.2010.07757.x.View ArticleGoogle Scholar
- Balasubramanian S, Neidle S: G-quadruplex nucleic acids as therapeutic targets. Curr Opin Chem Biol. 2009, 13: 345-53. 10.1016/j.cbpa.2009.04.637.View ArticleGoogle Scholar
- Pagano B, Martino L, Randazzo A, Giancola C: Stability and binding properties of a modified thrombin binding aptamer. Biophys J. 2008, 94: 562-569. 10.1529/biophysj.107.117382.View ArticleGoogle Scholar
- Griffin LC, Tidmarsh GF, Bock LC, Toole JJ, Leung LL: In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits. Blood. 1993, 81: 3271-3276.Google Scholar
- Padmanabhan K, Padmanabhan KP, Ferrara JD, Sadler JE, Tulinsky A: The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer. J Biol Chem. 1993, 268: 17651-17654.Google Scholar
- Paramasivan S, Rujan I, Bolton PH: Circular dichroism of quadruplex DNAs: applications to structure, cation effects and ligand binding. Methods. 2007, 43: 324-331. 10.1016/j.ymeth.2007.02.009.View ArticleGoogle Scholar
- Dapic V, Abdomerovic V, Marrington R, Peberdy J, Rodger A, Trent JO, Bates PJ: Biophysical and biological properties of quadruplex oligodeoxyribonucleotides. Nucleic Acids Res. 2003, 31: 2097-2107. 10.1093/nar/gkg316.View ArticleGoogle Scholar
- Kankia BI, Barany G, Musier-Forsyth K: Unfolding of DNA quadruplexes induced by HIV-1 nucleocapsid protein. Nucleic Acids Res. 2005, 33: 4395-4403. 10.1093/nar/gki741.View ArticleGoogle Scholar
- Kumar N, Maiti S: Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study. Biochem Biophys Res Commun. 2004, 319: 759-767. 10.1016/j.bbrc.2004.05.052.View ArticleGoogle Scholar
- Smirnov I, Shafer RH: Effect of loop sequence and size on DNA aptamer stability. Biochemistry. 2000, 39: 1462-1468. 10.1021/bi9919044.View ArticleGoogle Scholar
- Vorlickova M, Kejnovska I, Sagi J, Renciuk D, Bednarova K, Motlova J, Kypr J: Circular dichroism and guanine quadruplexes. Methods. 2012, 57: 64-75. 10.1016/j.ymeth.2012.03.011.View ArticleGoogle Scholar
- Bonifacio L, Church FC, Jarstfer MB: Effect of locked-nucleic acid on a biologically active g-quadruplex. A structure-activity relationship of the thrombin aptamer. Int J Mol Sci. 2008, 9: 422-433. 10.3390/ijms9030422.View ArticleGoogle Scholar
- Virno A, Randazzo A, Giancola C, Bucci M, Cirino G, Mayol L: A novel thrombin binding aptamer containing a G-LNA residue. Bioorg Med Chem. 2007, 15: 5710-5718. 10.1016/j.bmc.2007.06.008.View ArticleGoogle Scholar
- Pradhan D, Hansen LH, Vester B, Petersen M: Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K + solution. Chemistry. 2011, 17: 2405-2413. 10.1002/chem.201001961.View ArticleGoogle Scholar
- Georgiades SN, Abd Karim NH, Suntharalingam K, Vilar R: Interaction of metal complexes with G-quadruplex DNA. Angew Chem Int Ed Engl. 2010, 49: 4020-34. 10.1002/anie.200906363.View ArticleGoogle Scholar
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