Dual Aptamer-Functionalized 3D Plasmonic Metamolecule for Thrombin Sensing

DNA nanotechnology offers the possibility to rationally design structures with emergent properties by precisely controlling their geometry and functionality. Here, we demonstrate a DNA-based plasmonic metamolecule that is capable of sensing human thrombin proteins. The chiral reconfigurability of a DNA origami structure carrying two gold nanorods was used to provide optical read-out of thrombin binding through changes in the displayed plasmonic circular dichroism. In our experiments, each arm of the structure was modified with one of two different thrombin-binding aptamers—thrombin-binding aptamer (TBA) and HD22—in such a way that a thrombin molecule could be sandwiched by the aptamers to lock the metamolecule in a state of defined chirality. Our structure exhibited a Kd of 1.4 nM, which was an order of magnitude lower than those of the individual aptamers. The increased sensitivity arose from the avidity gained by the cooperative binding of the two aptamers, which was also reflected by a Hill coefficient of 1.3 ± 0.3. As we further exploited the strong plasmonic circular dichroism (CD) signals of the metamolecule, our method allowed one-step, high sensitivity optical detection of human thrombin proteins in solution.

[1]  E. Wang,et al.  G-quadruplex-based DNAzyme for facile colorimetric detection of thrombin. , 2008, Chemical communications.

[2]  Hao Yan,et al.  Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding. , 2008, Nature nanotechnology.

[3]  Jaroslav Kypr,et al.  Circular dichroism and conformational polymorphism of DNA , 2009, Nucleic acids research.

[4]  J. Szostak,et al.  Isolation of high-affinity GTP aptamers from partially structured RNA libraries , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Shawn M. Douglas,et al.  Folding DNA into Twisted and Curved Nanoscale Shapes , 2009, Science.

[6]  Masayuki Endo,et al.  DNA Origami Nanomachines , 2018, Molecules.

[7]  Tao Pan,et al.  A small metalloribozyme with a two-step mechanism , 1992, Nature.

[8]  E. Vermaas,et al.  Selection of single-stranded DNA molecules that bind and inhibit human thrombin , 1992, Nature.

[9]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[10]  J. Justin Gooding,et al.  Proximity extension of circular DNA aptamers with real-time protein detection , 2005, Nucleic acids research.

[11]  Koji Sode,et al.  Improvement of Aptamer Affinity by Dimerization , 2008, Sensors.

[12]  T. Heyduk,et al.  Nucleic acid-based fluorescence sensors for detecting proteins. , 2005, Analytical chemistry.

[13]  Veikko Linko,et al.  Dynamic DNA Origami Devices: from Strand-Displacement Reactions to External-Stimuli Responsive Systems , 2018, International journal of molecular sciences.

[14]  M F Kubik,et al.  Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. , 1997, Journal of molecular biology.

[15]  J. Szostak,et al.  Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures , 1992, Nature.

[16]  M Yarus,et al.  Diversity of oligonucleotide functions. , 1995, Annual review of biochemistry.

[17]  Timon Funck,et al.  Sensing Picomolar Concentrations of RNA Using Switchable Plasmonic Chirality. , 2018, Angewandte Chemie.

[18]  Juan Li,et al.  General colorimetric detection of proteins and small molecules based on cyclic enzymatic signal amplification and hairpin aptamer probe. , 2012, Analytical chemistry.

[19]  Yi Lu,et al.  A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity , 2007, Proceedings of the National Academy of Sciences.

[20]  M. Schilstra,et al.  Circular dichroism and its application to the study of biomolecules. , 2008, Methods in cell biology.

[21]  Tim Liedl,et al.  Molecular force spectroscopy with a DNA origami–based nanoscopic force clamp , 2016, Science.

[22]  J. Szostak,et al.  In vitro selection of functional nucleic acids. , 1999, Annual review of biochemistry.

[23]  Na Liu,et al.  Dynamic Plasmonic System That Responds to Thermal and Aptamer-Target Regulations. , 2018, Nano letters.

[24]  Juewen Liu,et al.  Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles. , 2005, Angewandte Chemie.

[25]  Mark Bathe,et al.  A primer to scaffolded DNA origami , 2011, Nature Methods.

[26]  Tim Liedl,et al.  DNA nanostructures in vitro, in vivo and on membranes , 2019, Nano Today.

[27]  D. Bunka,et al.  Aptamers come of age – at last , 2006, Nature Reviews Microbiology.

[28]  Joseph M Slocik,et al.  Theory of circular dichroism of nanomaterials comprising chiral molecules and nanocrystals: plasmon enhancement, dipole interactions, and dielectric effects. , 2010, Nano letters.

[29]  A. Kuzyk,et al.  Reconfigurable 3D plasmonic metamolecules. , 2014, Nature materials.

[30]  Ralf Jungmann,et al.  DNA Origami Route for Nanophotonics , 2018, ACS photonics.

[31]  Hao Yan,et al.  DNA Origami with Complex Curvatures in Three-Dimensional Space , 2011, Science.

[32]  P. Rothemund.  Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[33]  B. Sullenger,et al.  Aptamers: an emerging class of therapeutics. , 2005, Annual review of medicine.

[34]  Veikko Linko,et al.  On the Stability of DNA Origami Nanostructures in Low-Magnesium Buffers. , 2018, Angewandte Chemie.

[35]  M. Mascini,et al.  Aptamer-based biosensors for the detection of HIV-1 Tat protein. , 2005, Bioelectrochemistry.

[36]  Ronghua Yang,et al.  Noncovalent assembly of carbon nanotubes and single-stranded DNA: an effective sensing platform for probing biomolecular interactions. , 2008, Analytical chemistry.

[37]  Yi Lu,et al.  A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. , 2003, Journal of the American Chemical Society.

[38]  Yi Xiao,et al.  Aptamer-functionalized Au nanoparticles for the amplified optical detection of thrombin. , 2004, Journal of the American Chemical Society.

[39]  Luke P. Lee,et al.  Aptamer-based SERRS sensor for thrombin detection. , 2008, Nano letters.

[40]  N. Greenfield.  Using circular dichroism spectra to estimate protein secondary structure , 2007, Nature Protocols.

[41]  Robert J Gillies,et al.  Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: homooligomers of alpha-MSH. , 2004, Bioorganic & medicinal chemistry letters.

[42]  Lawrence A. Yannuzzi,et al.  PRECLINICAL AND PHASE 1A CLINICAL EVALUATION OF AN ANTI-VEGF PEGYLATED APTAMER (EYE001) FOR THE TREATMENT OF EXUDATIVE AGE-RELATED MACULAR DEGENERATION , 2002, Retina.

[43]  John I. Loewenstein,et al.  Anti-vascular endothelial growth factor therapy for subfoveal choroidal neovascularization secondary to age-related macular degeneration: phase II study results. , 2003, Ophthalmology.

[44]  X Chris Le,et al.  Aptamer-modified monolithic capillary chromatography for protein separation and detection. , 2008, Analytical chemistry.

[45]  K. Ikebukuro,et al.  Methods for Improving Aptamer Binding Affinity , 2016, Molecules.