Four steps for revealing and adjusting the 3D structure of aptamers in solution by small-angle X-ray scattering and computer simulation
- 118 Downloads
Nucleic acid (NA) aptamers bind to their targets with high affinity and selectivity. The three-dimensional (3D) structures of aptamers play a major role in these non-covalent interactions. Here, we use a four-step approach to determine a true 3D structure of aptamers in solution using small-angle X-ray scattering (SAXS) and molecular structure restoration (MSR). The approach consists of (i) acquiring SAXS experimental data of an aptamer in solution, (ii) building a spatial distribution of the molecule’s electron density using SAXS results, (iii) constructing a 3D model of the aptamer from its nucleotide primary sequence and secondary structure, and (iv) comparing and refining the modeled 3D structures with the experimental SAXS model. In the proof-of-principle we analyzed the 3D structure of RE31 aptamer to thrombin in a native free state at different temperatures and validated it by circular dichroism (CD). The resulting 3D structure of RE31 has the most energetically favorable conformation and the same elements such as a B-form duplex, non-complementary region, and two G-quartets which were previously reported by X-ray diffraction (XRD) from a single crystal. More broadly, this study demonstrates the complementary approach for constructing and adjusting the 3D structures of aptamers, DNAzymes, and ribozymes in solution, and could supply new opportunities for developing functional nucleic acids.
KeywordsAptamer Thrombin Three-dimensional structure Small-angle X-ray scattering Molecular modeling
Cryogenic electron microscopy
Fluorescence resonance energy transfer
Molecular structure restoration
Nuclear magnetic resonance spectroscopy
Small-angle X-ray scattering
Authors are grateful to Ana Gargaun for English grammar correction. This work was funded in parts by the Ministry of Science and Higher Education of the Russian Federation; project 0287-2019-0007 the Council of the President of the Russian Federation for Support of Young Scientists and Leading Scientific Schools (project no. SP-938.2015.5) and the grant of KSAI “Krasnoyarsk Regional Fund of Supporting Scientific and Technological Activities” for M.P., the internship “The study of the stacking of the secondary structure of DNA aptamers to thrombin” for R.M.
V.N. Zabluda, S.S. Zamay, S.G. Ovchinnikov created an idea, designed the overall concept, supervised the work. R. Moryachkov, M. Platunov, G. Peters, V.N. Zabluda, A. Melnichuk performed SAXS experiments, F.N. Tomilin, I. Shchugoreva, S.G. Ovchinnikov, A. Sokolov perform modeling, V. Spiridonova, A. Melnichuk, A. Atrokhova performed circular dichroic spectrum analysis and UV-melting, F.N. Tomilin, S.S. Zamay, G.S. Zamay, T.N. Zamay, M.V. Berezovski, R. Moryachkov, M. Platunov, A.S. Kichkailo analyzed all data and wrote the paper. All authors provided intellectual input, edited and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
- 6.Pang X, Cui C, Wan S, Jiang Y, Zhang L, Xia L, et al. Bioapplications of cell-SELEX-generated aptamers in cancer diagnostics, therapeutics, theranostics and biomarker discovery: a comprehensive review. Cancers (Basel). 2018;10(2). https://doi.org/10.3390/cancers10020047.
- 15.Kyogoku Y. NMR studies on structure and interaction of proteins and nucleic acids in solution. Tanpakushitsu Kakusan Koso. 1995;40(3):327–39.Google Scholar
- 17.van Buuren BN, Schleucher J, Wittmann V, Griesinger C, Schwalbe H, Wijmenga SS. NMR spectroscopic determination of the solution structure of a branched nucleic acid from residual dipolar couplings by using isotopically labeled nucleotides. Angew Chem Int Ed Engl. 2004;43(2):187–92. https://doi.org/10.1002/anie.200351632.CrossRefGoogle Scholar
- 21.Rambo RP, Tainer JA. Super-resolution in solution x-ray scattering and its applications to structural systems biology. Annu Rev Biophys. 2013;42:415–41. https://doi.org/10.1146/annurev-biophys-083012-130301.CrossRefGoogle Scholar
- 32.Guinier A. L'esprit de la recherche aux U. S. A. Atomes. 1947;2(20):378–82.Google Scholar
- 36.Stewart JJP. MOPAC2016. Stewart Computational Chemistry, Colorado Springs, CO, USA. http://openmopac.net/MOPAC2016.html. 2016
- 45.Russo Krauss I, Merlino A, Randazzo A, Novellino E, Mazzarella L, Sica F. High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity. Nucleic Acids Res. 2012;40(16):8119–28. https://doi.org/10.1093/nar/gks512.CrossRefGoogle Scholar
- 46.Spiridonova VA, Barinova KV, Glinkina KA, Melnichuk AV, Gainutdynov AA, Safenkova IV, et al. A family of DNA aptamers with varied duplex region length that forms complexes with thrombin and prothrombin. FEBS Lett. 2015;589(16):2043–9. https://doi.org/10.1016/j.febslet.2015.06.020.CrossRefGoogle Scholar