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Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely–used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule–biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled with small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR–biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor–groove–binding agent with DNA is evaluated by both kinetic and steady–state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule–DNA interaction is also presented.
Biological systems function on a platform of complex and integrated biomolecular interactions (1–3). Signaling, transcription control of gene expression and a host of other complex processes frequently are built on sets of sequential biomolecular interactions and subsequent reactions (4–8). These sequential processes that control and direct cellular functions can generally be understood in terms of the interaction of a small number of molecules in each step. Appropriate assembly of the entire sequential array gives the beautifully intricate mechanism of cell function. The entire process can be understood and explained on the basis of thermodynamic theory that has been firmly established for over 100 years. The biomolecular associations involve macromolecular complexes as well as small molecule-macromolecule interactions that are essential for the control of cell processes. Most drugs, for example, are small molecules that must interact with a macromolecular receptor in order to affect the target cell function in a selective manner (5–12). The field of chemical biology is built around design and use of small molecules to control specific aspects of cell function through biopolymer interactions (13–15). In order to understand this intricate array of interactions and control systems, it is very informative to evaluate all of the specific sequential steps that can be isolated. This quantitative interaction information is essential to put available structural results into the appropriate context of cell function.
To establish a basic quantitative characterization of the interactions it is essential to determine a set of basic thermodynamic quantities (16–21). Two questions must be answered at the start of the investigations: (i) what do we want to know about the interactions and (ii) how can we accurately and with a reasonable effort determine the desired information? In the optimum case the binding affinity (the equilibrium constant, K, and Gibbs energy of binding, ΔG), stoichiometry (n, the number of compounds bound to the biopolymer), cooperative effects in binding, and binding kinetics (the rate constants, k, that define the dynamics of the interaction) should be determined. These fundamental parameters are the keys to a molecular understanding of the interactions and how they affect cellular functions. To determine these parameters an accurate method of determining the concentration of each component that is not bound and the concentration of their complex is required. The information must be determined as a function of concentration at equilibrium for accurate K and n, and as a function of time for k. For each system then the question becomes one of how to accurately determine the necessary concentrations as a function of time, reactant total concentrations, solution conditions, temperature, etc. The interacting systems can range from as little as two molecules to as many as are required to form the final complex. As described above, a more complete understanding of the biomolecular complexes requires determination of additional thermodynamic quantities that characterize complex formation in detail.
Because biological molecules have widely different molecular characteristics that can and generally do change on complex formation, it can be difficult to find methods to evaluate the full array of interactions under an appropriate variety of conditions. This is the “reasonable effort” part of question (ii) above. For many biologically important interactions binding is very strong and experiments must be conducted at very low concentrations, down to the nanomolar or lower levels. In a binding experiment the compound concentration should vary from below to above the Kd in order to obtain accurate binding results and this requires significant concentrations of both free and complexed molecules (16,22–24). In many methods, for example, those that involve some separation of free and bound forms such as dialysis, the unbound concentration has to be determined and if this is below around 100 nM, as expected for the large K values generally observed in biological systems and as generally required for effective drug molecules, measurement can be difficult or impossible. These concentrations fall below the detection limit for many systems and may require special methods such as radiolabels or fluorescent probes for added sensitivity in detection. The labels may significantly increase the sensitivity of detection, however, they may also perturb the interaction that is being investigated. An attractive alternative method, which is operational down to very low concentrations, is the use of biosensors with surface plasmon resonance (SPR) detection (17,25–30). SPR responds to the refractive index or mass changes at the biospecific sensor surface on complex formation (27–32). Since the SPR signal responds directly to the amount of bound compound in real time, as versus indirect signals at equilibrium that are obtained for many physical measurements, it provides a very powerful method to study biomolecular interaction thermodynamics and kinetics. Use of the SPR signal and direct mass response to monitor biomolecular reactions also removes many difficulties with labeling or characterizing the diverse properties of biomolecules (25–31).
To illustrate the power of the SPR method, small organic cation interactions with specific DNA sequences will be used as examples. Small molecule targeting of DNA has applications in therapeutics, from cancer treatment to antiparasitic applications, and in biotechnology, and development of such molecules for control of nucleic acid function is at the heart of chemical biology (9,5–10,16,25,29,32–47). The interaction of nucleic acids with small molecules has been a primary area of interest and research since before the discovery of the double helical structure of DNA. There are many successful anticancer agents that intercalate or alkylate DNA and they can have quite varied structure and properties (34–37). Dicationic minor groove binding heterocycles which target eukaryotic organisms that cause parasitic diseases, such as malaria and sleeping sickness, have also been known for many years and are used in humans and animals (9,10,38–40). Therapeutic targeting of nucleic acids recently entered a new and very exciting area with the discovery that four–stranded, quadruplex DNA structures can occur in important cellular DNA regions from chromosomal telomeres to oncogene promoters (34,35,41–46). Several studies have clearly shown that interactions of a range of small molecules with quadruplexes can yield anticancer activity. Although this discussion focuses on DNA, essentially all of the methods can be applied to RNA interactions. Additional treatment of the biosensor surfaces may be required when using RNA, however, due to possible hydrolysis of RNA by a number of agents.
Applications with small molecules can be very challenging in terms of signal obtained on binding in a biosensor–SPR experiment. The larger the bound molecule, the larger the SPR signal and macromolecule binding can give large signal to noise ratios (27,29,31). We will show, however, that the biosensor–SPR method with a macromolecular sensor surface can be used under appropriate conditions to investigate small molecule binding with current state of the art instruments. Because of the varied properties of the compounds, it is difficult and time–consuming to find other suitable methods that can quantitatively define their interactions with DNA (or RNA) under a variety of conditions.
As described above, in biosensor–SPR instruments and experiments it is the bound compound on the surface that is detected by the change in plasmon resonance angle and this can be determined very accurately from a low to a high fraction of surface binding sites covered. The unbound or free solution compound concentration is not measured but is simply prepared by dilution and is in a constant concentration in the solution that flows over the sensor surface. Since the SPR signal responds to the refractive index or mass changes at the biospecific sensor surface on complex formation, no specific labeling of detection ability for the compounds is required (25–27,31). For compounds that have molecular weights of approximately 300 or more, the SPR technology provides a very attractive method to study interactions with nucleic acids or proteins immobilized to form a biospecific target surface.
For a single compound (C) binding to n equivalent sites on DNA (or other biopolymer) to give a complex (DNA•nCbound) the interaction process is described by kinetic and equilibrium equations as follows:
More complex models with nonequivalent sites are also well–known and are treated in a similar manner with more complex functions (27 and described below under Data Analysis, 3.2). Although a discussion of complex data fitting is beyond the scope of this article, the topic is covered in most biophysical chemistry texts and more complex models are included in commercial SPR instrument software packages as well as in software available from Myszka and coworkers (http://www.cores.utah.edu/interaction/software.html).
In the most common case it is of interest to study a variety of compounds for binding to a limited number of different DNAs. For this case, however, it is more convenient and efficient to immobilize the DNAs on the sensor surface to monitor complex formation. Biacore T100 and 2000/3000 instruments have sensorchips with four channels such that three DNAs can be immobilized with one flow cell left blank as a control for bulk refractive index subtraction. With a common sensor surface that has covalently attached streptavidin, a nucleic acid strand with biotin linked to either the 5′ or 3′ terminus can be captured to create the biospecific surface. The terminal attachment of biotin, through a flexible linker, leaves the nucleic acid binding sites open for complex formation. If streptavidin or the biotin linker creates problems with the interaction or nonspecific compound interactions, the nucleic acid can be synthesized with a terminal alkyl amine and the amine can be used to form a direct covalent amide bond with the surface through activation of carboxyl groups on the sensor surface. In Biacore instruments, which have been most widely used to date in the area of biosensor–SPR experiments, any molecule with free amino groups can be immobilized through amide bonds with activated carboxyl groups from sensor chip surface-linked carboxymethyl (CM) dextran. A range of other sensorchips surfaces and immobilization chemistries are also available and it is generally possible to find an appropriate surface for any biological interaction application (for Biacore instruments, see the web http://www.biacore.com/lifesciences/products/sensor_chips/guide/index.html).
The results from a biosensor–SPR experiment are typically presented as a set of sensorgrams, which plot a function that is directly related to the SPR angle versus time as shown in Figure 2. The angle change is reported in Biacore technology as resonance units (RU) where a 1000 RU response is equivalent to a change in surface concentration of about 1ng/mm2 of protein or DNA (the relationship between RU and ng of material bound will vary with the refractive index of the bound molecule) (27,29,31). With a DNA sequence immobilized on the chip surface, a compound solution is injected and as the solution flows over the surface, compound binding to the DNA is monitored by a change in SPR angle (as RU). After a selected time, buffer flow is restarted and dissociation of the complex can be monitored for an additional selected time period (Figure 2). Note that when the association phase collection time is long enough, a steady state plateau is reached such that the rate of binding of the small molecule equals the rate of dissociation of the complex and no change of signal with time is observed. The response of interest is the difference between the response in cells with immobilized DNA minus the response of the blank flow cell without DNA. If the added molecule does not bind to a target/receptor at the surface, the SPR angle change in the sample and reference flow cells will be the same in a properly functioning instrument and the signals, after subtraction, give a zero net RU response that is indicative of no binding. In the case where binding does occur, an extra amount, relative to the blank surface, of the added molecule is bound at the sensor surface and an additional SPR angle change is generated in the sample flow cell. Again, the amount of unbound compound in the flow solution is the same in the sample and reference flow cells and is subtracted so that only the bound molecule generates a positive SPR signal. The concentration of the unbound molecule is constant and is fixed by the concentration in the flow solution.
Both the association and dissociation phases of the sensorgram can be simultaneously fit to a desired binding model in several sensorgrams at different concentrations with a global fit routine (27–32). Global fitting allows the most accurate determination of the kinetics constants as well as calculation of the equilibrium constant, Ka, from the ratio of kinetic constants (Equation 1). It is also possible to determine Ka independently of rate constants by fitting the steady–state response versus the concentration of the binding molecule in the flow solution over a range of concentrations. For the binding process in Equation 1 the steady–state RU at each compound concentration is determined and Ka can be obtained by fitting to the following equation:
where RUmax is the maximum change in RU for binding to a single DNA site. It can be calculated, determined experimentally at high compound concentrations or used in the above equation as a fitting parameter such that Ka, n and RUmax are determined by fitting RU versus Cf. Note that the common term “r”, the moles of compound bound per mole of DNA, is equal to RU/RUmax. At high Cf where all binding sites are filled with compound, RU = RUmax · n.
The refractive index change in SPR experiments generates essentially the same response for each bound molecule and can provide a direct determination of the stoichiometry, n, in equations 1 and 2 provided that the amount of immobilized nucleic acid is known (29,31). If the complex dissociates slowly, the surface can be regenerated before complete dissociation has occurred by a solution that causes rapid dissociation of the complex without irreversible damage to the immobilized DNA (29,47). For example, a solution at low or high pH can unfold DNA and cause complex dissociation. It should be noted, however, that accurate fitting of the dissociation part of the curve requires collection of as much of the total dissociation signal as possible. Additional injections of buffer at pH near 7 allow the DNA to refold for the next binding experiment. If the immobilized DNA is composed of separate strands, the duplex must be reformed by hybridization.
After the dissociation/regeneration phase is over and a stable baseline is re–established, a second concentration sample can be injected to generate a second sensorgram. This process can be repeated with as many concentrations as needed to obtain a broad coverage of the fraction of compound bound to the nucleic acid site or sites (see Figure 2). The kinetic and equilibrium constants that describe the reaction in Equation 1 are obtained by global fitting the sensorgrams with equations from a kinetic model or by fitting steady–state RU versus concentration plots to an appropriate binding model. The models are the same for all types of binding experiments and are not unique to SPR methods. It should be emphasized that to obtain accurate kinetic information for a binding reaction, it is essential that the kinetics for transfer of the binding molecule to the surface immobilized nucleic acid (mass transfer) be faster than the binding reaction. Equilibrium information can be obtained, however, by fitting sensorgrams even when mass transfer limitations prevent accurate determination of kinetics (48). It should be emphasized that, when properly conducted, biosensor–SPR kinetic and equilibrium results are in excellent agreement with other methods (26,27,29,30,48–50).
These materials are for the Biacore T100, 3000 and 2000 research instruments but similar materials are required for other instruments.
The Biacore software supplied with the instruments allows users to write a method or to use a software wizard to set up experiments. Several important factors, such as flow rate, association time and dissociation time, injection order and surface regeneration, must be considered in setting up experiments. A sample method used to collect small molecule binding results on nucleic acid surfaces is shown below. The structure of the compound (DB293) and the biotin–labled DNA sequences (ATGA, AATT, ATAT) used in this example are shown in Figure 1.
We very much thank the NIH for funding the research that has made this review possible and the Georgia Research Alliance for funding of Biacore instruments. We also very much thank Professor David W. Boykin and his coworkers (Georgia State University, Atlanta, GA, USA) for supplying DB293 and many other DNA and RNA binding agents for SPR studies as well as for helpful discussions.
1Maintenance chips are available from GE Healthcare Inc. For desorb running, 50% DMSO and 10% DMSO can be used instead of Biacore desorb solution 1 and Biacore desorb solution 2 to effectively remove sticky small molecules. For biological samples such as protein, Sanitize method is also used after Desorb to insure that no microbial growth is present in the liquid injection and flow system.
2After running the regular Desorb, if the baseline is still not stable within ±1.0 RU/min (check with a new CM5 chip using running buffer), additional Super clean method may be used. First, run Desorb using 1% (v/v) acetic acid in place of Biacore desorb solution 1 and Biacore desorb solution 2, followed by one Prime to wash out the residual acetic acid. Then, run Desorb using 0.2 M sodium bicarbonate in place of SDS (solution 1) and glycine (solution 2), followed by one Prime to wash out the sodium bicarbonate residuals. Last, run Desorb using 6 M guanidine HCl for the SDS and 10 mM HCl for glycine. Prime the instrument a few times to thoroughly clean all residuals.
3Typically, an immobilization amount of 300–450 RUs of hairpin nucleic acid (~20–30 bases in length) is immobilized for running steady–state experiments and a low amount of 100–150 RUs for kinetic experiments to minimize mass transfer effects.
4A scrambled nonbinding sequence could be immobilized on flow cell 1 in some situations to provide a more similar control surface for subtraction.
5To visualize and illustrate a difference in stoichiometry for different nucleic acid hairpins, equal moles of nucleic acid can be immobilized on different flow cells using the equation (RU = C× moles bound × MWnucleic acid), where C is a relatively constant conversion factor for nucleic acid, proteins, etc. A higher level of response units (RU) is required for a higher molecular weight hairpin.
6Don’t open the package or dock the chip until the liposomes are prepared and ready for use to minimize adsorption of unwanted material from the air and running buffer on to the sensor chip.
7Detergents shoud not be used.
8Buffer degassing is very important and is particularly important if experiments are run at temperatures above 25 °C.
9For the Biaore 2000 instrument, even 10 times lower surfactant concentration (0.0005%, v/v polysorbate 20) can be applied to the running buffer and it works very well. While for Biacore 3000 and Biacore T100, higher concentration (0.005%, v/v polysorbate 20) is needed in the running buffer. Depending on the binding compound, a little higher binding affinity may be obtained when increasing surfactant concentration in the running buffer. Salt concentration can be adjusted based on the experimental requirement. The higher the salt concentration, the lower the binding affinity that will be obtained when cations bind to nucleic acids.
10If the small molecule requires the presence of a small amount of an organic solvent (e.g., <5% DMSO) to maintain solubility, the same amount of this organic solvent must be in the running buffer to minimize the refractive index difference.
11If the KA is unknown, it is necessary to conduct a preliminary experiment with several concentrations spread over a broad range to obtain an estimate of KA. A more focused set of concentrations is then prepared to cover the specific binding range.
12A short contact time, 30~60 s, is usually sufficient. Longer exposure to regeneration conditions involves greater risks for loss of binding activity on the surface, and often does not lead to improved regeneration.
13For the steady–state method, equilibrium, but not kinetics, constants can be obtained even when mass transfer effects dominate the observed kinetics. Thus, higher flow rates are not required in steady-state experiments as long as a clear steady–state plateau is obtained for determining RU at the steady–state. Higher flow rates (> 50 μl/min) are used for kinetic experiments to minimize mass transport effects.
14A sufficient association phase with a plateau region is needed for steady-state analysis. For the most accurate fitting of the dissociation phase it is best to allow sufficient time for the compound to dissociate at least 80% from the complex
15Many organic small molecules are easily adsorbed nonspecifically to the tubing of the injection micro–fluidics and are slowly released over the course of the experiment.
16Other software programs such as Scrubber2 and CLAMP are available for processing Biacore data. The results can also be exported and presented in graphing software such as KaleidaGraph for either PC or Macintosh computers. Although it is useful to experiment with different software packages, BIAevaluation (current version 4.1) is sufficient for most routine analyses of sensorgram data. For the Biacore T100 user, data processing can be performed automatically using the Biacore T100 evaluation software, which is much more convenient for new users.
17These two data processing steps are referred to as “double referencing”. Typically, multiple buffer injections are performed and averaged before subtraction. In double referencing, plots are made for each flow cell separately overlaying the control flow cell– corrected sensorgrams from buffer and all sample injections. The buffer sensorgram is then subtracted from the sample sensorgrams. “Double referencing” removes the systematic drifts and shifts in baseline and is helpful to minimize offset artifacts and also to correct the bulk shift that results from slight differences in injection buffer and running buffer (27).
18In some instances at low concentrations where the response does not reach the steady-state, the equilibrium responses can be obtained from kinetic fits of the sensorgrams utilizing the known RUmax from the higher concentration sensorgrams. This extrapolation method works well with sensorgrams where the observed response is at least 50% of the equilibrium RU.
19Various fitting models are included in the Biacore evaluation software and users can also write other models to be used with the program. While more complex binding models should only be used if the materials are pure, the experiment has been conducted properly, and the more complex model improves the fit significantly above the experimental error.
20In cases where a steady–state plateau is reached, the ratio of the rate constants (ka/kd) should be compared to the steady–state KA value. An agreement between the two methods suggests that the binding constant, KA, is correct but does not necessarily mean that the ka and kd values are correct due to possible mass transfer effects and possible correlation of constants (29,47).