Characterization of aptamer protein interactions using optical and acoustic biosensors

CHARACTERIZATION OF APTAMER-PROTEIN INTERACTIONS USING OPTICAL AND ACOUSTIC BIOSENSORS TANG QIANJUN (B.Eng Hunan University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements First of all, I would like to express my sincere gratitude to my supervisors, Associate Professor Loh Kian Ping from National University of Singapore (NUS) and Dr Su Xiaodi from Institute of Materials Research and Engineering (IMRE), for their kind guidance, support and encouragement throughout my research work I am thankful to all the current and former members of the groups from NUS and IMRE for their help and friendship I am also grateful to my family and friends for their invaluable love and support, without them, I can not go any further in my life Last but not least, my acknowledgement goes to NUS and IMRE for providing the financial support and the facilities to carry out the research work i Table of Contents Acknowledgement Table of Contents Summary List of Tables List of Figures List of Equations List of Symbols and Abbreviations Chapter Introduction 1.1 Biosensors 1.2 Thrombin and thrombin binding aptamers 1.3 G-quadruplex 1.4 Scope of this study Chapter Theory 2.1 Surface plasmon resonance 2.1.1 Maxwell equation of plane waves at interface 2.1.2 Surface plasmons at a metal/dielectric interface 2.1.3 Excitation of surface plasmons 2.1.4 SPR response to a thin film adsorption 2.2 Quartz crystal microbalance with dissipation monitoring 2.2.1 Piezoelectric excited acoustic waves and Sauerbrey equation 2.2.2 QCM liquid phase sensing 2.2.3 The dissipation factor 2.2.4 Modeling of QCM-D data Chapter Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to anti-thrombin DNA aptamers 3.1 Introduction 3.2 Experimental Section 3.2.1 Materials 3.2.2 SPR measurement 3.2.3 Sensor disk preparation 3.2.4 Assay procedures 3.3 Results and Discussion 3.3.1 A DNA spacer in TBA15 enhances thrombin binding capacity 3.3.2 Aptamer surface density affects thrombin binding capacity 3.3.3 Salt concentration affects specific thrombin-aptamer binding and nonspecific thrombin-DNA binding 3.3.4 Competition and displacement assay show that thrombin/TBA15-2 complex is more stable than thrombin/TBA15-1 complex 3.3.5 TBA15 and TBA29 bind to thrombin at two binding sites simultaneously 3.3.6 Thrombin can complex with immobilized TBA29 3.4 Conclusion Chapter Development of Colorimetric Assay for Studying Aptamer-thrombin-aptamer Sandwich Bindings i ii iv vi vii ix xi 1 10 10 10 13 15 17 19 19 21 23 24 28 28 28 28 30 32 34 36 36 37 39 41 43 46 48 49 ii 4.1 Introduction 4.2 Experimental Section 4.2.1 Materials 4.2.2 Mechanism of color reaction 4.2.3 Microwell plate-based colorimetric assay 4.2.4 SPR measurement 4.3 Results and Discussion 4.3.1 Construction of colorimetric assays 4.3.2 Optimization of assay conditions 4.3.3 The choice of primary aptamer 4.3.4 Spacer effects on sandwich complex efficiency 4.3.5 Spacer effects on detection sensitivity 4.3.6 TBA15–thrombin–TBA15 complex is formed at a lower efficiency 4.3.7 Thrombin quantification using sandwich colorimetric assay 4.4 Conclusion Chapter Quartz Crystal Microbalance Study of DNA G-quadruplex Folding and Thrombin-Aptamer Interaction 5.1 Introduction 5.2 Experimental Section 5.2.1 Materials 5.2.2 Sensor disk preparation 5.2.3 DNA immobilization and thrombin binding assay procedures 5.2.4 QCM-D and SPR measurement 5.2.5 Data modeling of aptamer film and aptamer-thrombin complex film 5.3 Results and Discussion 5.3.1 Salt concentration effects on DNA conformation 5.3.2 Modeling SPR and QCM-D data of G-quadruplex formation and thrombin-aptamer binding 5.3.3 Different binding behaviors and kinetics detected by QCM-D and SPR 5.3.4 QCM-D monitored aptamers-thrombin-aptamer sandwich complex formation 5.4 Conclusion BIBLIOGRAPHY 49 50 50 51 52 54 54 54 57 60 62 64 64 66 67 68 68 69 69 70 70 71 73 73 73 77 79 82 84 86 iii Summary Optical and acoustic biosensors have been successfully applied in the study of biomolecular interactions for several years In our study, surface plasmon resonance (SPR) spectroscopy, colorimetric assays and quartz crystal microbalance with dissipation monitoring (QCM-D) were employed to characterize the DNA Gquadruplex structure folding as well as the thrombin-aptamer interaction, in combination with different binding schemes (i.e one step binding, competition and displacement binding, sandwich binding plus a signaling step) All findings in this work would be invaluable in the study of DNA secondary structure and also the aptamer-protein interactions on surface, which in consequence will be useful in the development of DNA aptamer-based thrombin biosensors In chapter and 2, the general introduction of biosensor, thrombin, aptamer, Gqudrupelx as well as the underlying theories of SPR and QCM-D techniques are given In chapter 3, SPR spectroscopy was applied to study the interfacial binding characteristics of thrombin to its DNA aptamers on surface Using a 15-mer aptamer that binds thrombin primarily at the fibrinogen-recognition exosite as a model, respective effects of a DNA spacer, salt concentration, and aptamer surface density on thrombin binding capacity and stability were evaluated Immobilized 29-mer aptamer (specific to thrombin’s heparin-binding exosite) shows a lower affinity to thrombin than 15-mer aptamer, although it is known to have a higher affinity in solution phase Using a sandwiched assay scheme with the signaling step, we have observed the simultaneous binding of the 15-mer and 29-mer aptamers to thrombin at different exosites and found that one aptamer depletes thrombin’s affinity to the other when they bind together iv In chapter 4, colorimetric assays based on 96-well microplate were developed to study the formation of aptamer-thrombin-aptamer sandwich complexes on solid substrates A primary aptamer was first immobilized on streptavidin-modified microplate Thrombin was then applied to bind to the primary aptamer, followed by the addition of a secondary aptamer With the colorimetric assays, we have investigated: 1) the efficiency of sandwich complexes formed with different aptamers as primary aptamers, 2) the effects of DNA spacer in aptamers on sandwich complex formation and on detection sensitivity, and 3) the possibility of forming sandwich complex with two aptamers of the same sequence With an optimal sandwich design, thrombin quantification at nanomolar level was achieved In chapter 5, QCM-D was used to study the G-quadruplex DNA folding and thrombin-aptamer interactions, aiming to further understand the folding behavior on surface and the different binding kinetics detected by different instruments By comparing the different ΔD/Δf ratios and responses to salt concentration of DNA sequences with or without the ability to form G-quadruplex, we demonstrated the folding behavior on surface in situ By modeling the SPR and QCM data, the parameters of aptamer film and thrombin-aptamer complex film were obtained In addition, the kinetics of thrombin binding to aptamer immobilized on QCM chip and the formation of aptamer-thrombin-aptamer sandwich complex were studied and compared with the SPR results It shows that different sensing modes will give different apparent binding kinetics v List of Tables Table 3-1 DNA sequences used in this study 29 Table 4-1 Complex settings 57 Table 5-1 Buffer used in this study 70 Table 5-2 Summary of ΔD and Δf in different buffer conditions 76 Table 5-3 Analysis of the immobilized G-15 and S-15 DNA film in buffer using a Voigt-based viscoelastic model 78 Table 5-4 Analysis of the aptamer (G-15) and thrombin-aptamer complex in buffer using a Voigt-based viscoelastic model 79 vi List of Figures Figure 1-1 The ribbon diagram of human thrombinin complex with a DNA aptamer Figure 1-2 Schematic diagram of the G-quartet Figure 1-3 Different DNA quadruplex structures Figure 2-1 Schematic diagram of surface plasmon at the interface between a metal and a dielectric 10 Figure 2-2 The dispersion relation of free photos in a dielectric and in a coupling prism 16 Figure 2-3 Prism coupling geometries for Otto configuration and Kretschmann configuration 17 Figure 2-4 Typical surface plasmon resonance curves of the prism/gold/ethanol system 18 Figure 2-5 Picture of a sensor crystal 20 Figure 2-6 Schematic presentation of the model used to simulate a quartz crystal covered with a viscoelastic film and a bulk Newtonian liquid 25 Figure 3-1 Picture (A) and Schematic view (B) of the double-channel AutoLab SPR instrument setup 30 Figure 3-2 (A) Chemical structures of 1-mercapto-undecanole and 11-mercapto -(8-biotinamido-4, 7, dioxaoctyl-) undecanoylamide (B) Schematic diagram of the self-assembly process 33 Figure 3-3 Assay schemes used in this study 34 Figure 3-4 A DNA spacer in the immobilized aptamer enhances thrombin binding capacity 37 Figure 3-5 Thrombin/aptamer binding ratio is affected by aptamer surface density 38 Figure 3-6 Salt concentration effects on thrombin binding amount 40 Figure 3-7 Free aptamer competes for binding site with immobilized aptamers 41 Figure 3-8 Displacement assay 42 Figure 3-9 Formation of TBA15/thrombin/TBA29 sandwich complex 45 Figure 3-10 Thrombin binds TBA29 with lower capacities compared to TBA15-1 47 vii Figure 4-1 Schematic presentation of TMB oxidation 52 Figure 4-2 (a) A schematic illustration of the colorimetric assay (b) The secondary structure of the 15-mer and 29-mer TBA 53 Figure 4-3 SA-HRP nonspecific adsorption is measurable on various surface conditions 56 Figure 4-4 TWEEN 20 in washing buffer can improve the assay performance 58 Figure 4-5 BSA in SA-HRP binding buffer can block nonspecific SA-HRP adsorption and improve assay performance 59 Figure 4-6 SA-HRP concentration affects assay performance 59 Figure 4-7 OD values measured for the Complexes listed in Table and their corresponding controls 60 Figure 4-8 SPR response to the formation of sandwich complexes using either bTBA15 or bTBA29 as primary aptamer for immobilization 61 Figure 5-1 The Q-Sense D300 system overview 71 Figure 5-2 Setup for a batch/exchange mode measurement 72 Figure 5-3 Salt effect on immobilized DNA conformation change 74 Figure 5-4 (A) QCM-D measurements of the binding and displacement reactions outline (B) ΔD versus (-Δf) plots for the binding of thrombin to the G-15 aptamer immobilized surface 80 Figure 5-5 Kinetic plots of thrombin binding 81 Figure 5-6 f versus time at n=5 for the QCM-D data on aptamers-thrombin-aptamer sandwich complex formation 83 viii List of Equations Equation 2-1 11 Equation 2-2 11 Equation 2-3 11 Equation 2-4 11 Equation 2-5 11 Equation 2-6 12 Equation 2-7 12 Equation 2-8 12 Equation 2-9 13 Equation 2-10 13 Equation 2-11 13 Equation 2-12 13 Equation 2-13 14 Equation 2-14 14 Equation 2-15 14 Equation 2-16 14 Equation 2-17 15 Equation 2-18 15 Equation 2-19 15 Equation 2-20 18 Equation 2-21 20 Equation 2-22 21 Equation 2-23 22 Equation 2-24 22 ix (ΔmSPR) and the total mass of biomolecules and the trapped water in the biomolecular film obtained from QCM-D (ΔmVoigt) are listed in Table 5-3 and 5-4 The film thickness, effective film density and water content of G-15 and S-15 DNA films could be determined to give a quantitative estimation of the conformational differences (Table 5-3) It is found that the effective thickness of the G-15 DNA film is smaller than that of the S-15, which could be an evidence of the G-quadruplex structure formation of G-15 According to the modeled data in Table 5-3, the folded structure of G-15 has a shorter length (0.98 nm) than that of the randomly coiled single strand sequence S-15 (1.24 nm), which is consistent with the theoretical calculation of DNA persistence lengths in previous reports [108,109] In the earlier section, we have attributed the lower ΔD/Δf ratio of G-15 to the G-quadruplex formation The modeling results here again affirm the formation of intramolecular Gquadruplex structure of G-15 DNA by showing a smaller film thickness, higher effective film density and lower water content In comparison, the S-15 film has a lower film density due to more water content and a larger film thickness, which are also in line with the relative higher dissipation, i.e larger ΔD/Δf ratio ΔmSPR [ng/cm2] ΔmVoigt [ng/cm2] Effective film density (ρVoigt) Water content n=5/7 [g/dm ] (mass %) dVoigt [nm] n=5/7 G-15 40 116 1167 65 0.98 S-15 40 147 1133 71 1.24 Table 5-3 Analysis of the immobilized G-15 and S-15 DNA film in buffer using a Voigt-based viscoelastic model Since the G-15 DNA sequence is a thrombin binding aptamer, which possesses a high specificity and affinity to human α-thrombin, thrombin binding to G-15 78 immobilized surface was monitored Using Voigt-based viscoelastic model, the biophysical properties of the G-15 aptamer film and the subsequent thrombin-aptamer complex film are analyzed Modeled data are listed in Table 5-4, from which it can be observed that after thrombin binding the water content of the aptamer film decreased largely, from 65% to 28%, at the same time the effective film density increases from 1167 to 1422 ng/cm2, which together evidence the displacement of water when thrombin binds to aptamer The decreased ΔD/Δf ratio from aptamer film to thrombinaptamer complex film shows less energy dissipated in the latter, i.e the thrombinaptamer complex film is more rigid, which also indicates less water trapped in the protein-DNA complex film ∆D/∆f (1e-6/Hz) Effective film density (ρVoigt) Water content [g/dm3] (mass %) aptamer (G-15) 0.138 1167 65 thrombin-aptamer complex 0.046 1422 28 Table 5-4 Analysis of the aptamer (G-15) and thrombin-aptamer complex in buffer using a Voigt-based viscoelastic model 5.3.3 Different binding behaviors and kinetics detected by QCM-D and SPR In Figure 5-4 (A), it can be observed that D keeps on dropping during the time of thrombin incubation, which indicates that the overall biomolecular film gained rigidity upon thrombin binding This observation also affirms the previous conclusion from end point ΔD/Δf ratio decrease It could be attributed to the formation of a well structured complex between the DNA aptamer and thrombin, which displaces the water originally coupled within the DNA film 79 A 2.5 -15 -20 1.5 -25 D5 (1E-6) f 5/5 (Hz) 0.5 -30 20 40 60 80 Time (min) 1.05 ΔD (1E-6) B 0.95 0.85 0.75 27 27.5 28 28.5 29 29.5 -Δf (Hz) Figure 5-4 (A) QCM-D measurements of the binding and displacement reactions outline Point 1: Immobilization of G-15 Point 2: Binding of thrombin Point 3: Displacement by adding secondary aptamer The dashed ↑ arrows indicate the time when surface is rinsed with buffer (B) ΔD versus (-Δf) plots for the binding of thrombin to the G-15 aptamer immobilized surface In the ΔD-Δf plot (Figure 5-4 (B)), time is eliminated as an explicit parameter During the course of thrombin binding, D keeps on dropping even after f becomes stable It could be ascribed to the unique binding behavior detected by QCM-D, which is not only sensitive to the mass change, but also to the energy loss of the oscillatory system When Δf =0, i.e the total mass loading on the chip remains unchanged, there 80 still could be a dynamic mass exchange between thrombin protein and water inside the film In other words, the binding of thrombin (mass increase) to immobilized aptamer expels the water previously trapped inside the aptamer film (mass decrease) This exchanging procedure may result in net mass change equals to zero, which is corresponding to the stable stage of thrombin incubation step observed in SPR experiments But at the same time, it also could make the DNA film become more and more rigid as indicated by the constant decrease of the dissipation factor, which could not be reflected in SPR binding curve Normalized Response 1.2 0.8 0.6 0.4 QCM-D 0.2 SPR 0 10 15 20 Time (min) Figure 5-5 Kinetic plots of thrombin binding Δmass of QCM-D is calculated according to the Sauerbrey equation (Equation 2-22), Δmass of SPR is calculated according to the mass sensitivity factor of 120 mDeg per 100 ng/cm2 The saturated mass is set as 100% for both cases In order to compare the kinetics of thrombin-aptamer binding monitored by SPR and QCM-D, we normalize the binding signals measured from both instruments up to 20 incubation (Figure 5-5) For the interfacial thrombin binding on DNA aptamer of same surface coverage, and from a similar standstill liquid setup in both SPR and QCM systems, QCM kinetics appears to be faster than SPR kinetics The different apparent kinetics should be ascribed to the distinct sense modes of SPR and QCM 81 techniques SPR is more straightforwardly reflective of mass loading on surface over time, which corresponds to the continuous changes in refractive index of the interface; whereas, QCM-D measures mechanically coupled masses, including DNA, protein and water coupled Thus, the thrombin association on QCM-D chip appears to reach equilibrium faster With the SPR kinetics as reference, we believe QCM-D failed to reflect the real thrombin binding kinetics The change in the amount of trapped water complicates the QCM-D mass loading characteristics Although the protein may continuously binds to the DNA (as SPR curve shows), the mass increase may be compensated by the lost of water, making the overall mass remains unchanged 5.3.4 QCM-D monitored aptamer-thrombin-aptamer sandwich complex formation In association with the question as for what aptamer sequences are effectively involved in sandwich thrombin complex, thrombin binding to aptamer G-15 on the QCM-D chip surface is followed by addition of different secondary aptamers, G-15 or G-29 In Figure 5-6 (A), the assembly of primary G-15 on surface and the binding of thrombin on the immobilized G-15 show stepwise frequency drops, indicating the adsorption of molecules on QCM-D surface The subsequent addition of secondary G15 shows a direct frequency increase, indicating a mass decrease on the chip surface, which could be attributed to the dissociation of bound thrombin This characteristic is consist with previous SPR result [102], in which secondary G-15 addition resulted in mass decrease detectable as SPR angle decrease On the contrary, when the G-29 is used as the secondary aptamer, in Figure 5-6 (B), the frequency decreases during the binding, but increases slightly after rinse At the end point, Δf is negative, which means the final mass loading on the chip surface is increased From the previous 82 study [102], we know that the mass change of this sandwich system is a combinational result, including the dissociation of thrombin and the binding of the G29 aptamer A -5 G-15 f 5/5 (Hz) -10 thrombin -15 G-15 -20 SA -25 -30 -35 20 40 60 80 100 120 140 120 140 Time (min) B -10 G-15 f 5/5 (Hz) -20 thrombin -30 G-29 -40 SA -50 -60 20 40 60 80 100 Time (min) Figure 5-6 f versus time at n=5 for the QCM-D data on aptamers-thrombin-aptamer sandwich complex formation G-15 is first immobilized on the QCM-D chip, on to which thrombin (500 nM) is applied to bind At the end of thrombin binding, 500 nM G-15 (A) or G-29 (B) is applied After rinsing, SA (0.1 mg/ml) is added The dashed arrows indicate the time when the surface is rinsed with binding buffer According to the QCM-D result, at the first stage, the binding of G-29 and its coupling water dominates the mass change, which leads to a mass increase and frequency decrease Since the G-29 also has the displacement effect, after rinse it may take away more thrombin from the surface, which has a larger molecular weight (37 83 KDa) compared to G-29 DNA (9.5 KDa) itself, as a result, the loading mass will decrease and the corresponding frequency will increase This observation is unique in QCM-D, since SPR curve only showed a direct signal drop when adding the secondary aptamer [102] Again this should be ascribed to the different sensing modes of the two techniques Since SPR is only sensitive to the ‘dry mass’ on the surface, it could not detect the water coupled to the secondary aptamer As a result, the dissociation of the large thrombin molecular dominated the combinational effect and led a negative response (angle decrease) While the frequency change of QCM-D includes the effect of water coupled to the secondary aptamer, which could lead to a mass increase when the mass of thrombin dissociated is smaller than that of adsorbed secondary aptamer plus the coupled water In order to verify the presence of the secondary aptamer after the sandwich complex formation in both of the two cases, SA was introduced to recognize the biotin residue of the secondary aptamer After a ~20 SA incubation, Figure 5-6 (A) only shows a small f decrease, which may indicate a sandwich forming with low efficiency, while Figure 5-6 (B) shows a large f decrease, which indicates an obvious SA binding, i.e a more efficient aptamer-thrombin-aptamer sandwich formation This observation further confirmed the previous SPR results [102] 5.4 Conclusion By using QCM-D technique, we have determined the folding of G-quadruplex DNA on surface, with the scrambled DNA sequence as a reference The biophysical parameters of DNA film, such as effective film density, film thickness and also percentage of the water content, have been modeled These parameters are well reflective of the formation of G-quadruplex Moreover, as a thrombin aptamer, the 84 binding kinetics of G-15 to thrombin and formation of aptamer-thrombin-aptamer sandwich complex have also been investigated by using QCM-D, which are found to be distinguished from the SPR results due to the different sensing modes Furthermore, the biophysical parameters of the aptamer film before and after thrombin protein binding have also been documented, which provide new information about the binding event These findings would be invaluable for studying formation of DNA 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127 (2005) 12343 92 ... primary aptamers, 2) the effects of DNA spacer in aptamers on sandwich complex formation and on detection sensitivity, and 3) the possibility of forming sandwich complex with two aptamers of the... study of DNA secondary structure and also the aptamer- protein interactions on surface, which in consequence will be useful in the development of DNA aptamer- based thrombin biosensors In chapter and. .. SPR and QCM-D data of G-quadruplex formation and thrombin -aptamer binding 5.3.3 Different binding behaviors and kinetics detected by QCM-D and SPR 5.3.4 QCM-D monitored aptamers-thrombin-aptamer