Characterization of Aptamer Binding using SensíQ SPR Platforms APPLICATION NOTE INTRODUCTION Aptamers have the potential to provide a better solution in diagnostics and other research areas than traditional antibodies because of their stability, synthetic nature, and size. However, little is sometimes known about their analytical properties. Aptamer development has historically been limited to one-target at a time, generating high development costs. Base Pair Biotechnologies, Inc. (Houston TX) has addressed this severe limitation by successfully multiplexing the conventional aptamer selection process. Their platform technology is a patented approach to aptamer discovery that allows de novo aptamer discovery services at unprecedented cost, speed, and throughput. Demonstrated proficiency in aptamer development and experience with a variety of targets enables applications of their aptamers in a wide range of novel applications such as biosensor platforms. Eric Reese Ph.D. VP, Marketing Base Pair Biotechnologies aptamer discovery services include validation of aptamer binding by characterizing the aptamer target dissociation constant (K D ). Here we demonstrate the dissociation constant, K D, for two aptamers specific for Laminin and IgM mu chain protein targets developed by Base Pair Biotechnologies using the SensíQ, a dual-channel, semiautomated SPR system from SensíQ Technologies, Inc. (Oklahoma City, OK). WHY SENSIQ SPR INSTRUMENT PLATFORMS? Sensitive, low-cost platforms requiring minimal assay development to achieve high quality results. Directly measure nanomolar to millimolar binding constants. Affordable and sensitive semi-automated platform (SensiQ) ; sensitive to small molecules of ~250Da. FIGURE 1: SensiQ Semi-Automated Instrument
Using SensíQ s Pioneer instrument, the user can make decisions early on. Reliable affinity data (KD) and kinetic data (ka, kd) directly from a single injection with throughout of up to 384 samples per day or 768 samples automated. FIGURE 2: SensiQ Pioneer automated SPR platform WHY BASE PAIR BIOTECHNOLOGIES FOR YOUR APTAMER DEVELOPMENT Patented aptamer development platform decreasing cost and development time Limited risk discovery to complete Intellectual Property transfer Demonstrated success with a variety of protein, peptide, and small molecule targets Experience with various assay development with aptamer reagents Aptamers provided with analytical data to ensure specificity and with guaranteed affinity Proprietary project portal ensures communication with Base Pair scientists through the entire development process BACKGROUND Aptamer selection Aptamers are single-stranded DNA or RNA oligonucleotides selected to have unique threedimensional folding structure for binding to a variety of targets such as proteins, peptides, and even small molecules with affinity and specificity rivaling that of antibodies. They are typically selected in vitro by a process commonly referred to as SELEX [1,2] as depicted in FIGURE 3. Using a proprietary variant of this process, Base Pair Biotechnologies is developing aptamers to multiple targets simultaneously.
FIGURE 3 Overview of SELEX for Production of DNA Aptamers. A randomized library, flanked by two constant regions for PCR priming is constructed. The library is allowed to bind with the target and partitioned from the non-binding population. Following repeated rounds of selection and enrichment, high affinity DNA ligands are cloned and sequenced. Aptamer targets for this study Laminin is an extracellular matrix (ECM) multidomain trimeric glycoprotein and is the major non-collagenous component of basal lamina that supports adhesion, proliferation and differentiation.[3] IgM is by far the physically largest antibody in the human circulatory system. It is the first antibody to appear in response to initial exposure to antigen.[4] IgM heavy chain (mu chain) consists of 576 amino acids; there are five mu chains in each IgM molecule. IgM heavy chain is purified by reducing the disulfide bond connecting it to the IgM kappa and lambda light chains. Following several rounds of aptamer selection with these targets, aptamer clones were evaluated by SPR as described below. SPR In simplest terms, surface plasmon resonance (SPR) is a technique for detecting changes in refractive index at the surface of a sensor. The sensor is comprised of a glass substrate and thin gold coating. Light passes through the substrate and is reflected off of the gold coating (FIGURE 4). At certain angles of incidence, a portion of the light energy couples through the gold coating and creates a surface plasmon wave at the sample and gold surface interface. The angle of incident light required to sustain the surface plasmon wave is very sensitive to changes in refractive index at the surface (due to mass change), and it is these changes that are used to monitor the association and dissociation of biomolecules. A detector array measures the reflected light and calculates the angle at which light has coupled through the gold surface as an attenuation or dip in the signal. Changes in the signal dip location on the array are monitored and recorded as refractive index increases (mass binding) or decreases (mass dissociating). By repeating this measurement at different analyte concentrations, calculations can be made for k a, k d, and K D.
FIGURE 4-BASIC SPR PRINCIPLE RESULTS Biotinylated Laminin specific aptamer was immobilized to the NeutrAvidin modified COOH5 chip (SensíQ Technologies) followed by biotin quenching. Then different concentrations of the Laminin target (Sigma L2020) were applied to confirm binding and also to get the K D for the aptamer target interaction. From the table below (TABLE 1), the average K D = 5.0 ±0.9 nm. This affinity compares very well to that of a typical monoclonal antibody. Kinetics analysis using SensiQ s Qdat software: Non-linear regression, pseudo-first order 1:1 interaction FIGURE 5. Overlaid fit of association and dissociation results of Laminin aptamer binding with Laminin protein concentrations of 600, 120, 25 nm; For this assay, this almost perfect overlaid fit with the 1:1 binding shows that the binding assay conditions were appropriate.
k a (M -1 s -1 ) k d (s -1 ) Rmax (RU) K D Laminin protein 1.16 ±0.01e5 6 ±1e-4 255.3 ±0.5 5.0 ±0.9 nm TABLE 1. Kinetics table showing rate and affinity constants obtained for Laminin aptamer binding to Laminin protein using the SensíQ system. Similar to the Laminin experiment, biotinylated IgM mu specific aptamer was immobilized and then different concentrations of the IgM target (Athens Research 16-16-090713-MU) were applied to check the binding and also to get the K D value of the aptamer IgM interaction. From the table below (TABLE 2), K D = 178 ±6 pm. Kinetics analysis using SensiQ s Qdat software: Non-linear regression, pseudo-first order 1:1 interaction FIGURE 6. Overlaid fit of association and dissociation results of IgM mu chain aptamer binding with IgM protein concentrations 41, 20.5, 10.3, 5.15, 1.25 nm; IgM mu k a (M -1 s -1 ) k d (s -1 ) Rmax (RU) K D 1.82 ±0.02e7 3.2 ±0.1e-3 52.3 ±0.1 178 ±5 pm TABLE 2: Kinetics table showing rate and affinity constants obtained for IgM aptamer binding to IgM mu protein using the SensíQ system.
CONCLUSION The SensiQ semi-automated SPR platform has been demonstrated to provide binding affinity and kinetics for aptamers developed and produced by Base Pair Technologies, determining average K D = 5.0 ±0.9 nm for Laminin as target and average K D = 178 ±6 pm for IgM mu chain as target. The SensiQ is a an affordable and sensitive semi-automated 2 channel SPR platform designed for small molecule interaction analysis, while Base Pair Technologies provides quality aptamers with guaranteed specificity and affinity. SensíQ Technologies also offers the SensiQ Pioneer SPR platform, a quantum leap in realtime, label-free biomolecular interaction analysis. Pioneer utilizes the advantages of evolved surface chemistries and state-of-the-art data analysis tools to provide kinetic, affinity, and concentration data researchers can apply with confidence. With superior versatility, full automation, and ease of use in an affordable, high quality surface plasmon resonance system, Pioneer will have a definite impact on your research endeavors. SensiQ Technologies and Base Pair Technologies, a winning combination in the development and characterization of aptamers. For information about custom aptamer development: Base Pair Biotechnologies, Inc. 8058 El Rio St. Houston, TX 77054 (281) 829-8876 info@basepairbio.com REFERENCES 11 Tuerk C, Gold L: Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249:505-10. 2. Ellington AD, Szostak JW: In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346:818-22. 3. http://www.sigmaaldrich.com/catalog/product/sigma/l2020?lang=en®ion=us 4. Houghton Mifflin Company, 2004. "Immunoglobulin M." The American Heritage Dictionary of the English Language, Fourth Edition. Accessed on 12 Oct. 2007