Minseon Cho

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.

Dual-aptamer-based delivery vehicle of doxorubicin to both PSMA (+) and PSMA (-) prostate cancers.

We have designed a dual-aptamer complex specific to both prostate-specific membrane antigens (PSMA) (+) and (-) prostate cancer cells. In the complex, an A10 RNA aptamer targeting PSMA (+) cells and a DUP-1 peptide aptamer specific to PSMA (-) cells were conjugated through streptavidin. Doxorubicin-loaded onto the stem region of the A10 aptamer was delivered not only to PSMA (+) cells but to PSMA (-) cells, and eventually induced apoptosis in both types of prostate cancer cells. Cell death was monitored by measuring guanine concentration in cells using differential pulse voltammetry (DPV), a simple and rapid electrochemical method, and was further confirmed by directly observing cell morphologies cultured on the transparent indium tin oxide (ITO) glass electrode and checking their viabilities using a trypan blue assay. To investigate the in vivo application of the dual-aptamer system, both A10 and DUP-1 aptamers were immobilized on the surface of thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION). Selective cell uptakes and effective drug delivery action of these probes were verified by Prussian blue staining and trypan blue staining, respectively.

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.

Simultaneous electrochemical detection of both PSMA (+) and PSMA (-) prostate cancer cells using an RNA/peptide dual-aptamer probe

Using an RNA/peptide dual-aptamer probe, both PSMA (+) and PSMA (-) prostate cancer cells were simultaneously detected by electrochemical impedance spectroscopy. This approach can be applied as a general tool for early diagnosis of prostate cancer.

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.

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.

Array-based Discovery of Aptamer Pairs

Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site “bidentate” target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant “hot spot” epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.

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.

Abstract 2227: Aptamer selection for cancer markers: High-throughput, quantitative selection and characterization of nucleic acid aptamers for human angiopoietin-2.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Abstract Nucleic acid-based aptamers represent a promising class of affinity reagents, as they can be chemically synthesized and modified, undergo reversible folding and exhibit excellent stability. Thus, the identification of aptamers that bind to specific cancer markers may serve as a powerful tool for early diagnosis and treatments of cancers. Unfortunately, conventional methods of aptamer generation remain inherently low-throughput in nature, and there is a pressing need for alternative technologies that can generate high quality aptamers in a high-throughput and economical manner. Towards the goal of accelerating aptamer discovery, we have developed the Quantitative Parallel Aptamer Selection System (QPASS) - an integrated platform that combines microfluidic selection, next-generation sequencing and in situ-synthesized aptamer arrays for parallel binding measurements. As a model, we used QPASS to select aptamers that specifically bind to human angiopoietin-2, an important mediator of angiogenesis and a biomarker of colon, prostate and breast cancers; after microfluidic selection and sequencing, we fabricated a custom aptamer array containing 120,000 sequences representing the most enriched aptamer candidates. Using this array, we simultaneously obtained equilibrium dissociation constants (Kd) and binding specificity data for these candidate aptamers in a massively parallel manner. Furthermore, we show that our array can operate directly in undiluted serum, enabling rapid identification of aptamers that perform optimally in complex mixtures. By exploiting the scalability and commercial availability of high-throughput sequencing and nucleic acid arrays, the QPASS platform offers the capability to simultaneously analyze and characterize unprecedented numbers of aptamer candidates—either against a common cancer marker, or for screening in parallel against multiple different biomarkers. Citation Format: Minseon Cho, Seung Soo Oh, Jeff Nie, Ron Stewart, Michael Eisenstein, James Chambers, Jamey D. Marth, James A. Thomson, Tom H. Soh. Aptamer selection for cancer markers: High-throughput, quantitative selection and characterization of nucleic acid aptamers for human angiopoietin-2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2013-2227