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ForteBio Interactions Newsletter Biosensor photo

SPRING 2012    VOLUME 5    ISSUE 1

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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, high-selectivity ligands produced in vitro by a process commonly known as SELEX. While the selection of DNA and RNA aptamers has been described for some time, the SELEX process was traditionally performed against a single target at a time requiring weeks to months for successful execution. We have developed a proprietary process for multiplexing SELEX to discover aptamers against multiple targets simultaneously, thereby greatly increasing the throughput of the process. Our aptamer discovery services include validation of aptamer binding by characterizing the aptamer:target dissociation constant (kd) before delivery of aptamer materials to the customer for further testing. With an increasing customer base and a longer list of targets, it becomes increasingly important to implement higher throughput methods for aptamer validation. The ForteBio Octet platform provides a fast, simple, and cost-effective means to characterize multiple aptamer clones at a throughput that meets our customers’ demands.

INTRO TO APTAMERS

Aptamers are single-stranded DNA or RNA oligonucleotides selected to have unique three-dimensional folding structures 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. The initial randomized library applied to an immobilized target comprises approximately 1015 unique oligonucleotide sequences of 30–40 bases in length bracketed by constant regions for PCR priming. The output of the process is therefore several (5–20) clonal DNA sequences likely to have specific affinity for the target molecule. As mentioned above, a quantitative validation of the binding of such clones is of critical importance to our process and business.

GENERAL WORKFLOW

In general, we can take two approaches to affinity characterization of our aptamer products — either immobilizing the aptamer or the target itself. Each approach may have specific advantages and disadvantages. Immobilization of the aptamer itself allows a modular approach in which each of our aptamers is treated identically. In other words, there are no protein-specific immobilization conditions to optimize. This approach has the disadvantage, however, of requiring additional protein material when offering multiple protein analyte concentrations to the biosensor. Immobilization of the protein itself requires less total protein, but immobilization (primarily through lysine residues) may be perturbing to protein epitopes and may require protein-to-protein optimization depending on the nature of the target. Ultimately, both approaches are complementary and we therefore present facile protocols for each below.

METHOD 1: APTAMER IMMOBILIZATION

We have developed two methods for ready aptamer immobilization to ForteBio’s Dip and Read biosensors:

  • Direct immobilization of biotinylated DNA on streptavidin biosensors.
  • Biotinylated polyA capture method of aptamer immobilization

In the first method of aptamer immobilization, a DNA clone is appended at the 5′- or 3′- end and offered directly to ForteBio’s Streptavidin Biosensor. For most aptamers we have observed minimal perturbation or detrimental effect on binding due to such immobilization. Indeed, we can choose aptamer clones based on secondary structure that should be minimally affected by tethering at either end.

Kinetic assay set up for direct immobilization of biotinylated DNA to streptavidin biosensors.

FIGURE 1: Kinetic assay set up for direct immobilization of biotinylated DNA to streptavidin biosensors.

Drop. Read. Done! BLItz is simple to use.

FIGURE 2: Processed kinetic data for 1 µM #387 hCG aptamer-biotin and hCG protein analyte showing overlaid fits with KD = 56.6 nM.

 

Kinetic assay set up for immobilization of aptamer with biotinylated polyA approach.

FIGURE 3: Kinetic assay set up for immobilization of aptamer with biotinylated polyA approach.

 

 

In the second method of aptamer immobilization, a DNA aptamer clone is appended with a 14-mer poly-thymidine sequence at the 5′-end. This allows for hybridization to a biosensor pretreated with a biotin-polyA. The advantage of this approach is further steric spacing of the aptamer from the biosensor surface and, again, a modular approach to biosensor preparation. Finally, this configuration exactly matches some of our customers’ bead-based applications, allowing for large batches of polyA beads to be prepared before aptamer-specific functionalization.

Direct Immobilization of Biotinylated DNA to Streptavidin Biosensors (Single Reference Well) on Octet RED96 System

 

Sample Plate Preparation
  1. Prepare a 96-well plate with required ligands, analytes and wash buffers.
  2. All steps in the following example (Figure 1–2) were performed with a shake speed of 1000 rpm. Optimal shake speeds may vary.
Prepare and Load Biosensor Surface with Biotinylated Aptamer
  1. Place the biosensors in the SELEX buffer and equilibrate for 1 minute.
  2. Load the biosensors with 1 µM biotinylated hCG aptamer for 10 minutes.
  3. Perform a wash/baseline step in SELEX buffer for 1 minute.
Assess Association with Analyte Target Protein
  1. After the baseline step, place the biosensors in a dilution series of hCG protein for 10 minutes. For some analytes with a fast binding rate, even 3–5 minutes of association time will be enough to perform kinetic analysis.
  2. Place the biosensors in the SELEX buffer for 10 minutes for dissociation. If inter-step correction is required, the same well used for baseline should also be used for dissociation.

Processed kinetic data

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.

 

 

Kinetic assay set up for immobilization of protein to AR2G biosensors

FIGURE 5: Kinetic assay set up for immobilization of protein to AR2G biosensors.

 

Biotinylated PolyA Capture Method for Aptamer Immobilization (Single Reference Well) on Octet RED96 System

 

Sample Plate Preparation
  1. Prepare a 96-well plate with required ligands, analytes and wash buffers.
  2. All steps in the following example (Figures 3–4) were performed with a shake speed of 1000 rpm. Optimal shake speeds may vary.
Prepare and Load Biosensor Surface with Biotinylated PolyA Oligo and PolyT Aptamer
  1. Place the biosensors in the SELEX buffer and equilibrate for 1 minute.
  2. Block the biosensor surfaces with 1% NFDM for 300 seconds.
  3. Wash the biosensors in SELEX buffer for ~600 seconds to ensure unbound NFDM is removed.
  4. Load the biosensors with 1 µM 5′ biotinylated polyA oligo for 10 minutes.
  5. Perform a wash/baseline step in SELEX buffer.
  6. Load the biosensors with 10 µM Hb aptamer having 3′14T, for 10 minutes.
  7. Wash the biosensors with SELEX buffer.
Assess Association with Analyte Target Protein
  1. After the baseline step, place the biosensors in a dilution series of Hb protein for 10 minutes. For analytes with a fast binding rate, even 3–5 minutes of association time will be enough to perform kinetic analysis.
  2. Place the biosensors in SELEX buffer for 10 minutes for dissociation. If inter-step correction is required, the same well used for baseline should also be used for dissociation.

Data Processing

The data obtained is processed to determine the overlaid fits and the Kd, kon and koff values.

The reference well is subtracted from the analyte wells for buffer artifacts. Then y-axis alignment, inter-step correction and Savitzky-Golay filtering are also applied to the data.

The processed data is then allowed to fit a curve for association and dissociation using 1:1 model fitting with either global or local fitting.

METHOD 2: PROTEIN IMMOBILIZATION

For protein immobilization we use ForteBio’s Dip and Read AR2G biosensors. In this particular example, hsp27, a protein implicated in breast cancer3, was covalently immobilized to amine-reactive second generation (AR2G) biosensors using standard EDC/NHS coupling. Four different concentrations were immobilized on different biosensors. A novel biotinylated aptamer clone selected by Base Pair Biotechnologies was then applied to each biosensor at 1 µM. To obtain additional signal, the biosensors were then briefly washed in buffer and subsequently dipped in an equal concentration of streptavidin-HRP conjugate.

Quantitating the streptavidin–HRP step of kinetics assay. 1:2000 streptavidin-HRP conjugate by known concentration (µg/mL)

FIGURE 6: Quantitating the streptavidin–HRP step of kinetics assay. 1:2000 streptavidin-HRP conjugate by known concentration (µg/mL).

Standard curve of log hsp27 concentration vs. streptavidin-HRP binding rate.

FIGURE 7: Standard curve of log hsp27 concentration vs. streptavidin-HRP binding rate.

 

Protein Immobilization on AR2G Biosensors on Octet RED96 System

Sample Plate Preparation
  1. Prepare a 96-well plate with required ligands, analytes and wash buffers.
  2. All steps in the following example (Figures 5–7) were performed at a shake speed of 1000 rpm. Optimal shake speeds may vary.
Prepare and Load Biosensor Surface with Protein
  1. Place the biosensors in water and equilibrate for 30 seconds.
  2. Load 20 mM EDC and 10 mM NHS for 300 seconds.
  3. Load a dilution series of hsp27 protein (0.2 µM, 2 nM, 20 pM, 0.2 pM) in 10 mM acetate buffer, pH 6.0, for 1200 seconds. Since the amount of protein loading is low, the loading time is increased.
  4. Quench the remaining unreacted surface with 1 M Tris, pH 8.0, for 300 seconds.
  5. Move the biosensors into SELEX running buffer (20 mM Tris, 100 mM NaCl, 0.005% Tween20, pH 7.4) for 1800 seconds.
Associate Aptamer to the Immobilized Protein
  1. Load 1 µM hsp27 aptamer (Oligo #410) with 3’biotin for 300 seconds
Assess Association with Streptavidin-HRP and TMB Substrate
  1. After loading aptamer, add 1:2000 streptavidin-HRP enzyme conjugate for 1200 seconds.
  2. Load the final step, 200 µL 1X TMB substrate, in each of the wells for 1200 seconds.

Data Processing

Taking the initial slope of the response (ForteBio’s usual approach) results in a log-log standard curve. As can be seen in Figure 7, the linear fit is quite good (R2 = 0.96). Good signal-to-noise was obtained at an hsp27 concentration of 0.2 pM which compares favorably with even the best ELISAs. For additional signal and sensitivity, we can readily add a standard HRP substrate.

CONCLUSION

In a short amount of time we have adapted our laboratory workflow from surface plasmon resonance (SPR) to BLI analysis with considerable satisfaction. Because the ForteBio instrument does not employ a flow cell, we do not experience the instrument down time associated with microfluidic clogging, etc. Additionally, the use of largely modular workflows requiring little protein-to-protein optimization have allowed us to address increasing customer demand for our aptamer discovery services.

References:

  1. 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, 346:818–822.
  3. Kang SH, Kang KW, Kim K-H, Kwon B, Kim S-K, Lee H-Y, Kong S-Y, Lee ES, Jang S-G, Yoo BC: Upregulated HSP27 in human breast cancer cells reduces Herceptin susceptibility by increasing Her2 protein stability. BMC Cancer 2008, 8:286.

Download Application Note 5 for more experimental details, reagent requirements and operational tips at www.fortebio.com/literature.html