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SPR for Aptamer-Molecule Interactions SPR f SP R for or Ap Aptam tamer er-Based Based Mol Molecular ecular In Interactions eractions in in Programmable Mat Programmable Materi erials als Reichert Technologies Webinar September 22, 2015 Erin Gaddes The Wang Lab: Biomolecular & Biomimetic Materials The Pennsylvania State University University Park, PA 16802

SPR for Aptamer-Molecule Interactions Outline • Introduction to surface plasmon resonance (SPR) • What is it? How does it work? • Samples and detection strategies • Data analysis • SPR for analysis of oligo-biomolecule interactions • Cell Capture and Release • Growth factor loading and release • Signal amplification

SPR for Aptamer-Molecule Interactions Surface Plasmon Resonance Technique used to examine molecular interactions in real time • Sensor chip contains Wang lab SPR setup: Reichert SR7500DC dual immobilized ligand channel system • Microfluidic system delivers analyte • Optical measurement system : changes in local refractive index � changes in mass at sensor chip-solution Daghestani & Day . Sensors 2010, 10, 9630-9646. interface

SPR for Aptamer-Molecule Interactions Optical Detection n 2 • Light entering the prism above the critical angle is totally internally reflected ϴ SPR • These photons produce an n 1 evanescent wave at surface interface • Mobile electrons of metal surface treated as plasma n 1 > n 2 • Surface plasmons , from density ϴ SPR > ϴ Critical fluctuations at the interface, propagate along the interface

SPR for Aptamer-Molecule Interactions Optical Detection • When momentum of photons matches that of surface plasmons, resonance occurs, based on: • Light angle • Wavelength • Refractive indices of materials • Photons excite the plasmons � reduction in detected light • This reduction occurs as resonance angle is approached

SPR for Aptamer-Molecule Interactions Optical detection • Light illuminated on surface at range of angles • Output determines angle of minimum reflectivity • Mass changes at interface alter local refractive index � alter resonance angle http://www.reichertspr.co m/

SPR for Aptamer-Molecule Interactions Sensor Chip • Sensor chip consists of glass coated with a thin metal layer • Metal functionalized for immobilization of ligand • Amine coupling (EDC/NHS) • Streptavidin/neutravidin- biotin • Gold-thiol • Polymer matrix for balance between binding sites and N.J. de Mol, M.J.E. Fischer (eds.), Surface http://www.reichertspr.co Plasmon Resonance, Methods in Molecular signal strength m/ Biology, 2010.

SPR for Aptamer-Molecule Interactions Flow Cell • Two channels • Immobilization of ligand on sample channel • Reference channel with no ligand • Both channels treated with analyte for binding analysis Jahanshahi et al. Scientific Reports 2014, 4, 3851. http://www.reichertspr.co m/

SPR for Aptamer-Molecule Interactions Data Analysis • Kinetic analysis software (TraceDrawer) http://www.reichertspr.co m/ • Alignment for start time, response • Blank subtractions • Kinetic model fitting • 1:1 • 2:1, 1:2 • Mass transport N.J. de Mol, M.J.E. Fischer (eds.), Surface Plasmon Resonance, Methods in Molecular depletion considerations Biology, 2010.

SPR for Aptamer-Molecule Interactions SPR Data • Binding kinetics Stephenson-Brown et al. Analyst 2013, 138, 7140- 7145. • Equilibrium analysis • Binding specificity • Molecular interactions • Protein • Small molecules • Cells Le et al. Analytica Chimica • Oligonucleotides Acta 2013, 761, 143-148.

SPR for Aptamer-Molecule Interactions Aptamers • Nucleic acid aptamers: • Short, single-stranded oligonucleotides • Selected from randomized libraries • Interact with biomolecules (e.g. surface receptors) • Merits • Robust • High throughput chemical synthesis • Little batch-to-batch variation • No significant immunogenicity http://www.amsbio.com/ 2013

SPR for Aptamer-Molecule Interactions SPR for Oligonucleotide Interactions • Biosensors • Sequence specificity • Hybridization • DNA polymers • Competitive displacement • Aptamer affinity • VEGF • Thrombin Chen et al. Biosensors and Hasegawa et al. Sensors 2008, 8, 1090-1098. Bioelectronics 2014, 61, 83-87. • PTK7 receptors

SPR for Aptamer-Molecule Interactions B + Scrambled Sequence Specificity C B + C (20 base pairs) B + C (25 base pairs) Scrambled B Sequence Sequence 350 C + B C 300 B + + 250 ∆µRIU 200 B binds A C displaces B Sequence + 150 A with 20 from A by 100 forming 25 immobilize base pairs d on chip base pairs 50 A 0 0 500 1000 1500 2000 Time [s] Zhang et al. JACS 2012, 134, 15716-15719.

SPR for Aptamer-Molecule Interactions Application: Tumor Cell Capture and Release Complementary Sequence Release Scrambled Functional Hydrogel regeneration Cell Aptamer Release Display Cell Catch Zhang et al. JACS 2012, 134, 15716-15719.

SPR for Aptamer-Molecule Interactions Aptamer Length vs Affinity 10mer 9mer 8mer 7mer 6mer 10mer + trigger 10mer + control 180 180 150 150 120 120 Δ μ RIU Δ μ RIU 90 90 60 60 30 30 0 0 0 200 400 600 0 200 400 600 Injection time [s] Injection time [s] Zhang et al. Chemical Communications 2013, 49, 9600- 9602.

SPR for Aptamer-Molecule Interactions Aptamer Affinity Evaluation via SPR High Affinity 300 100 nM Response Aptamer 200 [µRIU] Comparison of Anti-PDGF 50 nM BB Aptamers at 100 nM 100 25 nM 250 0 Response [µRIU] 0 250 500 Time [s] 200 High Moderate Affinity 100 nM 150 Response 200 Aptamer 50 nM [µRIU] 25 nM 100 Moderat 100 12.5 nM e 6.25 nM 50 0 Low 3.125 nM 0 250 500 0 Time [s] 250 0 250 500 Low Affinity 100 nM Response 200 Time [s] 50 nM [µRIU] Aptamer 150 25 nM 100 12.5 nM 50 6.25 nM 0 3.125 nM 0 250 500 Battig et al. Biomaterials 2014, 35, 8040-8048. Time [s]

SPR for Aptamer-Molecule Interactions Application: Loading and Release of Growth Factors Battig et al. Biomaterials 2014, 35, 8040-8048.

SPR for Aptamer-Molecule Interactions SPR Examination of DNA Polymerization DI is immobilized on sensor chip DM 1 + 1600 DM 2 1400 1200 1000 Δ μ RIU 800 DM 1 + DNA DI 600 DM 2 Polymer 400 DM 1 200 DM 2 0 0 1000 2000 3000 4000 Time [s] Chen et al. Small 2013, 23, 3944-3949.

SPR for Aptamer-Molecule Interactions Application: DNA Polymer Nanoparticles for Signal Amplification Chen et al. Small 2013, 23, 3944-3949.

SPR for Aptamer-Molecule Interactions Formation of Polyvalent Aptamers Richards et al. Biomacromolecules 2014, 15, 4561- 4569.

SPR for Aptamer-Molecule Interactions Application: Polyvalent Aptamers for Tumor Cell Capture Richards et al. Biomacromolecules 2014, 15, 4561- 4569.

SPR for Aptamer-Molecule Interactions Polyvalent Aptamer Triggered Depolymerization Gaddes et al . Biomacromolecules 2015, 16, 1382- 1389.

SPR for Aptamer-Molecule Interactions Summary • SPR is a technique to detect molecular interactions in real time via alterations in local refractive index • Used to determine binding specificity, kinetics, and molecular interactions between proteins, nucleic acids, cells, and other small molecules • Nucleic acid aptamers have ability to bind targets with tunable affinity • SPR utilized to evaluate aptamer specificity, DNA hybridization, and triggered dissociation

SPR for Aptamer-Molecule Interactions Resources • Mol, Nico J. de, and Marcel J. E. Fischer, eds. Surface Plasmon Resonance Methods and Protocols . New York, NY: Humana Press, 2010. • Wong, Chi Lok, and Malini Olivo . “Surface Plasmon Resonance Imaging Sensors: A Review.” Plasmonics 9.4 (2014): 809 – 824. • Reichert Technologies: http://www.reichertspr.com/ • TraceDrawer: http://www.ridgeview.eu/software/tracedrawer/ • Scrubber: http://www.biologic.com.au/

SPR for Aptamer-Molecule Interactions Acknowledgements • Dr. Yong Wang • The Wang Lab • Dr. Mark R. Battig • Dr. Niancao Chen • Shihui Li • Reichert Technologies Funding: National Science Foundation (DMR • 1322332) Pennsylvania State College of Engineering • http://www.mri.psu.edu/about/millennium-science- Pennsylvania State Materials Research • Institute complex.asp