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Routine Characterization of DNA Aptamer Affinity to Recombinant Protein Targets Ilavarasi Gandhi, Research Assistant, Base Pair Biotechnologies, Inc., George W

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Routine Characterization of DNA Aptamer Affinity to Recombinant Protein Targets Ilavarasi Gandhi, Research Assistant, Base Pair Biotechnologies, Inc., George W InteractIons • sPrInG 2012 Routine Characterization of DNA Aptamer Affinity to Recombinant Protein Targets Ilavarasi Gandhi, Research Assistant, Base Pair Biotechnologies, Inc., George W. Jackson, Ph.D., Founder and Chief Scientist, Base Pair Biotechnologies, Inc. Nucleic acid aptamers are high affinity, ing to a variety of targets such as proteins, tion of the protein itself requires less total high-selectivity ligands produced in vitro by a peptides, and even small molecules with affin- protein, but immobilization (primarily through process commonly known as SELEX . While the ity and specificity rivaling that of antibodies . lysine residues) may be perturbing to protein selection of DNA and RNA aptamers has been They are typically selected in vitro by a process epitopes and may require protein-to-protein described for some time, the SELEX process commonly referred to as SELEX 1, 2 . The initial optimization depending on the nature of was traditionally performed against a single randomized library applied to an immobilized the target . Ultimately, both approaches are target at a time requiring weeks to months target comprises approximately 1015 unique complementary and we therefore present for successful execution . We have developed oligonucleotide sequences of 30–40 bases in facile protocols for each below . a proprietary process for multiplexing SELEX length bracketed by constant regions for PCR to discover aptamers against multiple targets priming . The output of the process is therefore METHOD 1: APTAMER simultaneously, thereby greatly increasing the several (5–20) clonal DNA sequences likely to IMMOBILIZATION throughput of the process . Our aptamer dis- have specific affinity for the target molecule . covery services include validation of aptamer As mentioned above, a quantitative valida- We have developed two methods for ready binding by characterizing the aptamer:target tion of the binding of such clones is of critical aptamer immobilization to ForteBio’s Dip and Read biosensors: dissociation constant (kd) before delivery of importance to our process and business . aptamer materials to the customer for further • Direct immobilization of biotinylated DNA testing . With an increasing customer base and GENERAL WORKFLOW on streptavidin biosensors . a longer list of targets, it becomes increasingly In general, we can take two approaches to important to implement higher throughput • Biotinylated polyA capture method of affinity characterization of our aptamer prod- methods for aptamer validation . The ForteBio aptamer immobilization ucts — either immobilizing the aptamer or the Octet platform provides a fast, simple, and target itself . Each approach may have specific In the first method of aptamer immobiliza- cost-effective means to characterize multiple advantages and disadvantages . Immobiliza- tion, a DNA clone is appended at the 5'- or aptamer clones at a throughput that meets tion of the aptamer itself allows a modular 3'-end and offered directly to ForteBio’s our customers’ demands . approach in which each of our aptamers is Streptavidin Biosensor . For most aptamers treated identically . In other words, there are no we have observed minimal perturbation or Intro to Aptamers protein-specific immobilization conditions to detrimental effect on binding due to such im- optimize . This approach has the disadvantage, mobilization . Indeed, we can choose aptamer Aptamers are single-stranded DNA or RNA however, of requiring additional protein mate- clones based on secondary structure that oligonucleotides selected to have unique rial when offering multiple protein analyte should be minimally affected by tethering at three-dimensional folding structures for bind- concentrations to the biosensor . Immobiliza- either end . 0.14 BaselineSELEX in buer 1μM hCGaptamer-biotin BaselineSELEX in buer hCG portein Dissociationin SELEX buer 0.13 0.10 0.12 0.05 0.11 0.00 0.10 -0.05 0.09 -0.10 -0.15 0.08 -0.20 0.07 -0.25 0.06 Binding, nm Binding, nm -0.30 0.05 -0.35 A9 0.04 B9 -0.40 C9 0.03 -0.45 D9 0.02 E9 -0.50 0.01 -0.55 0.00 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 0 100 200 300 400 500 600 700 800 900 1,000 1,100 1,200 T ime (sec) T ime (sec) FIGURE 1: Kinetic assay set up for direct immobilization of FIGURE 2: Processed kinetic data for 1 µM #387 hCG aptamer-biotin and hCG biotinylated DNA to streptavidin biosensors . protein analyte showing overlaid fits with KD = 56 .6 nM . 4 InteractIons • sPrInG 2012 Direct Immobilization of Biotinylated DNA to Streptavidin Biosensors (Single Reference Well) on Octet RED96 System Blocking with 1% NFDM Baseline in SELEX buffer 0.60 Baseline in SELEX buffer 1 μM 5’biotin polyA Baseline in SELEX buffer 10 μM Hb aptamer with 3’14T Baseline in SELEX buffer Association with Hb protein Dissociation in SELEX buffer Sample Plate Preparation 1 Prepare a 96-well plate with required 0.40 ligands, analytes and wash buffers . 0.20 2 All steps in the following example (Figures 1–2) were performed with a 0.00 shake speed of 1000 rpm . Optimal shake speeds may vary . -0.20 Prepare and Load Biosensor Surface with -0.40 Biotinylated Aptamer Binding, nm 1 Place the biosensors in the SELEX buffer -0.60 and equilibrate for 1 minute . -0.80 2 Load the biosensors with 1 μM biotinyl- A5 B5 ated hCG aptamer for 10 minutes . -1.00 D5 3 Perform a wash/baseline step in SELEX buffer for 1 minute . -1.20 0 600 1,200 1,800 2,400 3,000 3,600 4,200 4,800 5,400 T ime (sec) Assess Association with Analyte Target FIGURE 3: Kinetic assay set up for immobilization of aptamer with biotinylated polyA approach . Protein 1 After the baseline step, place the biosensors in a dilution series of hCG In the second method of aptamer immobi- biosensor preparation . Finally, this configura- protein for 10 minutes . For some analytes lization, a DNA aptamer clone is appended tion exactly matches some of our customers’ with a fast binding rate, even 3–5 minutes with a 14-mer poly-thymidine sequence at bead-based applications, allowing for large of association time will be enough to the 5'-end . This allows for hybridization to a batches of polyA beads to be prepared before perform kinetic analysis . biosensor pretreated with a biotin-polyA . The aptamer-specific functionalization . 2 Place the biosensors in the SELEX buffer advantage of this approach is further steric for 10 minutes for dissociation . If inter- spacing of the aptamer from the biosensor step correction is required, the same well surface and, again, a modular approach to used for baseline should also be used for dissociation . A B 0.80 0.80 0.70 0.70 0.60 0.60 0.50 0.50 0.40 0.40 Binding, nm Binding, nm 0.30 0.30 0.20 0.20 1:1 binding 1:2 (bivalent K = 25.6 nM analyte) binding 0.10 D 0.10 KD = 12.3 nm 0.00 0.00 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 Time (sec) T ime (sec) FIGURE 4: Processed kinetic data showing overlaid fit and KD values . A) Load 1: 1 µM load of 5'biotin-polyA (14A) . Load 2: 10 µM 3'14T gly Hb aptamer 72H09 #439 . Non-gly Hb protein at 1 and 0 .5 µM analyte . B) Load 1: 5'biotin-polyA . Load 2: 3'14T #439 Hb aptamer 72 H09 . Hb protein analyte . 5 InteractIons • sPrInG 2012 Equilibratein water EDC/NHSActivation LoadingHsp27 Protein Quench1 withM Tris, pH 8.0 Wash inSELEX buer AssociationHsp27 with Aptamer LoadingStreptavidin 1:2000 HRP LoadingSubstrate 1X TMB 24 20 16 12 8 4 Binding, nm 0 -4 -8 -12 0 300 600 900 1,200 1,500 1,800 2,100 2,400 2,700 3,000 3,300 3,600 3,900 4,200 4,500 4,800 5,100 5,400 5,700 6,000 6,300 T ime (sec) FIGURE 5: Kinetic assay set up for immobilization of protein to AR2G biosensors . Biotinylated PolyA Capture Method 5 Perform a wash/baseline step in SELEX The reference well is subtracted from the for Aptamer Immobilization (Single buffer . analyte wells for buffer artifacts . Then y-axis Reference Well) on Octet RED96 System alignment, inter-step correction and Savitzky- 6 Load the biosensors with 10 μM Hb ap- Golay filtering are also applied to the data . Sample Plate Preparation tamer having 3'14T, for 10 minutes . 1 Prepare a 96-well plate with required 7 Wash the biosensors with SELEX buffer . The processed data is then allowed to fit a curve ligands, analytes and wash buffers . for association and dissociation using 1:1 model Assess Association with Analyte Target fitting with either global or local fitting . 2 All steps in the following example Protein (Figures 3–4) were performed with a shake speed of 1000 rpm . Optimal shake 1 After the baseline step, place the biosen- METHOD 2: PROTEIN speeds may vary . sors in a dilution series of Hb protein for IMMOBILIZATION 10 minutes . For analytes with a fast bind- For protein immobilization we use Forte- ing rate, even 3–5 minutes of association Prepare and Load Biosensor Surface with Bio’s Dip and Read AR2G biosensors . In time will be enough to perform kinetic Biotinylated PolyA Oligo and PolyT Aptamer this particular example, hsp27, a protein analysis . 1 Place the biosensors in the SELEX buffer implicated in breast cancer3, was covalently and equilibrate for 1 minute . 2 Place the biosensors in SELEX buffer for 10 immobilized to amine-reactive second gen- minutes for dissociation . If inter-step correc- 2 Block the biosensor surfaces with 1% eration (AR2G) biosensors using standard tion is required, the same well used for base- NFDM for 300 seconds .
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