Andrew T. Csordas

Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing

We describe the integration of microfluidic selection with high-throughput DNA sequencing technology for rapid and efficient discovery of nucleic acid aptamers. The Quantitative Selection of Aptamers through Sequencing method tracks the copy number and enrichment-fold of more than 10 million individual sequences through multiple selection rounds, enabling the identification of high-affinity aptamers without the need for the pool to fully converge to a small number of sequences. Importantly, this method allows the discrimination of sequences that arise from experimental biases rather than true high-affinity target binding. As a demonstration, we have identified aptamers that specifically bind to PDGF-BB protein with Kd < 3 nM within 3 rounds. Furthermore, we show that the aptamers identified by Quantitative Selection of Aptamers through Sequencing have ∼3–8-fold higher affinity and ∼2–4-fold higher specificity relative to those discovered through conventional cloning methods. Given that many biocombinatorial libraries are encoded with nucleic acids, we extrapolate that our method may be extended to other types of libraries for a range of molecular functions.

Selection of phage-displayed peptides on live adherent cells in microfluidic channels

Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.

Detection of proteins in serum by micromagnetic aptamer PCR (MAP) technology.

The effective diagnosis and prognosis of many diseases depends on the ability to quantitatively measure protein biomarkers from clinical samples at low concentrations. For example, fluctuations in serum concentrations of cytokines, such as platelet-derived growth factor-BB (PDGF-BB), can serve as indicators of tumor angiogenesis, whereas levels of virus-related proteins such as hemagglutinin can indicate progression of an infection. Accurate detection of diagnostic biomarkers in blood is often challenging because of its complex composition comprising thousands of proteins with concentrations ranging over 12 orders of magnitude. 5] Albumin, for example, constitutes approximately half of the total serum protein (30–50 mgmL ), while many important disease-related biomarkers exist at concentrations as low as 1 pg mL . 5] Enzyme-linked immunosorbent assay (ELISA) is a standard approach to detect protein biomarkers directly from blood. Unfortunately, this assay can suffer from a lengthy development period for specific antibodies, insufficient sensitivity, limited dynamic range, 8, 9] and long assay times involving multiple washing steps, thereby limiting its usefulness and making it impractical to implement at the point of care. Several groups have developed innovative approaches to improve the sensitivity and dynamic range of ELISAs by combining antibody-based molecular recognition with nucleic acid amplification-based detection, such as proximity ligation, immuno-PCR, and bio-barcode detection. However, because interferents in blood can inhibit many amplification reactions, there is a critical need for universal sample preparation systems that allow amplification-based detection of protein biomarkers from complex samples in a monolithic, disposable, and automated format. Herein, we report the micromagnetic aptamer PCR (MAP) detection system, which integrates high-gradient magnetic field sample preparation in a microfluidic device with aptamer-based real-time PCR readout, to achieve highly sensitive and quantitative detection of protein targets directly from complex samples. As a model, we demonstrate the capability to quantitatively detect the cancer biomarker PDGF-BB over a wide dynamic range (62 fm to 1 nm) in a complex background of serum with clearly discernable and reproducible PCR amplification signals. The detection assay starts with the incubation of a serum sample containing PDGF-BB target protein with magnetic beads coated with capture antibody and anti-PDGF PCR aptamers, which incorporate flanking PCR primer sequences (Figure 1A). As with ELISA, the use of dual-affinity reagents significantly increases the specificity of detection. After the incubation step, the sample was loaded into a micromagnetic separation (MMS) chip, in which magnetically labeled antibody–target–aptamer complexes were trapped by the high local magnetic field gradients generated by microfabricated ferromagnetic structures (MFSs) patterned within the microchannel. Meanwhile, nontarget serum proteins, unused reagents, and PCR contaminants were continuously washed out during separation (Figure 1B). After washing the trapped beads, the external magnetic field was removed, which demagnetized the MFSs thereby allowing magnetic target complexes to be eluted with phosphate-buffered saline containing 0.25 mm MgCl2 (PBSM; Figure 1C). The entire separation and purification process (trapping, washing, and bead release) required about 30 min. One microliter of collected eluent was directly subjected to real-time PCR analysis, which yielded a signal proportional to the concentration of target protein in the serum sample (Figure 1D). Note that real-time PCR calibration curves with and without magnetic beads verified that the presence of 1 10 antibodycoated magnetic beads per PCR reaction volume did not [*] S. S. Oh, Dr. Y. Xiao, Prof. H. T. Soh Materials Department, Department of Mechanical Engineering University of California, Santa Barbara Santa Barbara, CA 93106 (USA) E-mail: yixiao@physics.ucsb.edu tsoh@engr.ucsb.edu Dr. A. Csordas Institute for Collaborative Biotechnologies University of California, Santa Barbara (USA) Dr. A. E. Gerdon Department of Chemistry, Emmanuel College, Boston (USA)

Detection of Telomerase Activity in High Concentration of Cell Lysates Using Primer-Modified Gold Nanoparticles

Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.

Rapid and Label-Free Strategy to Isolate Aptamers for Metal Ions

Generating aptamers that bind to specific metal ions is challenging because existing aptamer discovery methods typically require chemical labels or modifications that can alter the structure and properties of the ions. In this work, we report an aptamer discovery method that enables us to generate high-quality structure-switching aptamers (SSAs) that undergo a conformational change upon binding a metal ion target, without the requirement of labels or chemical modifications. Our method is more efficient than conventional selection methods because it enables direct measurement of target binding via fluorescence-activated cell sorting (FACS), isolating only the desired aptamers with the highest affinity. Using this strategy, we obtained a highly specific DNA SSA with ∼30-fold higher affinity than the best aptamer for Hg(2+) in the literature. We also discovered DNA aptamers that bind to Cu(2+) with excellent affinity and specificity. Both aptamers were obtained within four rounds of screening, demonstrating the efficiency of our aptamer discovery method. Given the growing availability of FACS, we believe our method offers a general strategy for discovering high-quality aptamers for other ions and small-molecule targets in an efficient and reproducible manner.

Measurement of Aptamer–Protein Interactions with Back-Scattering Interferometry

We report the quantitative measurement of aptamer-protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (K(d)) of 3.84 nM (Tasset-thrombin) and 5.96 nM (Bock-thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the K(d) of Bock aptamer binding to preformed Tasset-thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.

Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics

Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.