Seung Soo Oh

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)

Quantitative selection and parallel characterization of aptamers

Significance The comprehensive functional mapping of the human proteome will require access to high-quality affinity reagents that specifically bind to their respective proteins with high affinities. Unfortunately, currently available antibodies can only target a small fraction of the proteome, and their affinity and specificity can vary considerably for each protein. Thus there is an urgent need for novel technologies capable of generating alternative, synthetic affinity reagents in a scalable and economical manner. Toward this end, we report a unique screening system (termed the “Quantitative Parallel Aptamer Selection System”) that can accelerate discovery of high-quality aptamer reagents by enabling simultaneous measurements of binding affinity (Kd) and specificity for thousands of aptamers in parallel. Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and next-generation sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (Kd) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with Kd as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure−function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding.

In vitro selection of structure-switching, self-reporting aptamers

We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.

Probing the limits of aptamer affinity with a microfluidic SELEX platform.

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (Kd) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a Kd of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer Kd.

Controlled delivery of DNA origami on patterned surfaces

Due to its capacity for programmable self-assembly, wellestablished modes of chemical synthesis, and exceptional stability, DNA serves as a powerful nanoscale structural material. In particular, the recent invention of DNA origami technology has established a paradigm in which DNA’s capacity for deterministic self-assembly into essentially any discrete two-dimensional (2D) shape can be exploited for the construction of molecular ‘‘bread boards’’. For example, previous groups have demonstrated the delivery of nanoparticles to specific positions within an origami scaffold with nanometer-scale precision and the weaving of DNA aptamers into origami for the assembly of protein arrays. However, in order to fully harness the potential of DNA as a universal nanoscale template, it is important not only to control the position of cargo material within the origami scaffold but also to accurately control the position and orientation of the

Synthetic Aptamer-Polymer Hybrid Constructs for Programmed Drug Delivery into Specific Target Cells

Viruses have evolved specialized mechanisms to efficiently transport nucleic acids and other biomolecules into specific host cells. They achieve this by performing a coordinated series of complex functions, resulting in delivery that is far more efficient than existing synthetic delivery mechanisms. Inspired by these natural systems, we describe a process for synthesizing chemically defined molecular constructs that likewise achieve targeted delivery through a series of coordinated functions. We employ an efficient “click chemistry” technique to synthesize aptamer-polymer hybrids (APHs), coupling cell-targeting aptamers to block copolymers that secure a therapeutic payload in an inactive state. Upon recognizing the targeted cell-surface marker, the APH enters the host cell via endocytosis, at which point the payload is triggered to be released into the cytoplasm. After visualizing this process with coumarin dye, we demonstrate targeted killing of tumor cells with doxorubicin. Importantly, this process can be generalized to yield APHs that specifically target different surface markers.

Improving Aptamer Selection Efficiency through Volume Dilution, Magnetic Concentration, and Continuous Washing in Microfluidic Channels

The generation of nucleic acid aptamers with high affinity typically entails a time-consuming, iterative process of binding, separation, and amplification. It would therefore be beneficial to develop an efficient selection strategy that can generate these high-quality aptamers rapidly, economically, and reproducibly. Toward this goal, we have developed a method that efficiently generates DNA aptamers with slow off-rates. This methodology, called VDC-MSELEX, pairs the volume dilution challenge process with microfluidic separation for magnetic bead-assisted aptamer selection. This method offers improved aptamer selection efficiencies through the application of highly stringent selection conditions: it retrieves a small number (<10(6)) of magnetic beads suspended in a large volume (>50 mL) and concentrates them into a microfluidic chamber (8 μL) with minimal loss for continuous washing. We performed three rounds of the VDC-MSELEX using streptavidin (SA) as the target and obtained new DNA aptamer sequences with low nanomolar affinity that specifically bind to the SA proteins.

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.