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Multiplexed In Vitro Selection Of Aptamers Using Nanoporous Sol-Gel Arrays With Integrated Microheaters

Seung-min Park1, Abdullah Ozer2, Kylan Szeto1, Raymond Wu2, Jiyoung Ahn3, Minjoung Jo3, Dong-ki Lee4, Soyoun Kim3, John T. Lis2, Moonsoo Jin5, and Harold G. Craighead1

1. School of Applied and Engineering Physics, 2. Department of Molecular Biology and Genetics, 5. Department of Biomedical Engineering, Cornell University, Ithaca, NY

3. Department of Biomedical Engineering, Dongguk University, South Korea

4. Department of Chemistry, Sungkyunkwan University, South Korea

Research Summary

Aptamers have emerged as alternative protein capture reagents that, like antibodies, show high affinity and specificity for their targets. Aptamers often have affinities in the picomolar to low nanomolar range. They can fold into unique 3D structures that make specific contacts with a target protein over a large surface area. Unlike antibodies, generation of protein-specific aptamers is not limited by the immunogenicity or the toxicity of the protein. Once specific aptamers are sequenced, unlimited amounts of the same aptamer can be synthesized with a little effort. However, what is lacking is the means to generate aptamers rapidly to multiple targets in a high throughput and cost-effective way.

Figure 1: The schematics of multiplex microfluidic SELEX. Aptamers in randomized nucleic acid (NA) pool will bind target proteins confined in sol-gel droplets on the microfluidic device, protein-bound aptamers will be thermally eluted by integrated aluminum electrodes. Then, selected aptamers are either re-amplified and used for the next round of SELEX or identified by massively parallel sequencing.

Aptamers are selected against targets using an in vitro selection technique known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) as in Fig 1. With the conventional methods, each of the multiple cycles of SELEX requires ~2 days to perform and selections are usually performed with only one or a few protein targets. Recently, significant advancement has been made in automating SELEX to reduce some of the handling, cost, and duration of the selection process; however, selection of aptamers for multiple targets in parallel has not been fully developed. Also, laborious cloning and sequencing of individual aptamers have remained as the final steps of both manual and small-scale, automated SELEX methods.

Figure 2: View of the actual 24 microfluidic SELEX device and its sub-structures. The left image shows the fully functional microfluidic SELEX device with a PDMS cover. Each protein immobilized nanoporous sol-gel (middle, right images) has an individually addressable microheater.


Figure 3: Work flow of microfluidic SELEX. Several TBP aptamers were isolated after the 11th round of conventional filter binding. Our microfluidic SELEX method required fewer cycles of SELEX than that of the conventional SELEX method. The microfluidic SELEX was performed after two rounds of conventional SELEX. Final aptamer products from two experiments were sequenced and aligned. Our microfluidic SELEX system improved selection efficiency by as much as 50 %.

Significant progress was made in developing small-scale prototypes of a microfluidic platform that functions very well relative to the conventional filter binding SELEX [1]. Our current methodology utilizes nanoporous silica-based sol-gel droplets to confine different target proteins in microfluidic chambers that are arrayed at specific positions along a micro-channel. Sol-gel is not only suitable for immobilization of large amounts of proteins in a 3-dimensional environment, but also allows protein-nucleic acid interactions [2]. In this study, we created a SELEX-on-a-chip device harboring an array of sol-gel droplets with separate microheaters built underneath each droplet for multiplex binding and partitioning steps of the SELEX procedure. Sol-gel droplets allow the competitive binding of nucleic acid aptamers to different target proteins in each droplet, while the microheaters provides local heating to disrupt aptamer-protein interaction and allow selective elution of aptamers from each droplet. After elution, selected aptamers are individually reamplified then either combined into a pool and subjected to another round of selection or sequenced for identification.

Our microfluidic SELEX system shown in Fig. 2 improved selection efficiency, reducing the number of selection cycles needed to produce high-affinity aptamers by as much as 50% when compared to conventional filter-binding SELEX (Fig. 3). We selected aptamers that bind tightly and specifically, that were identical or homologous to those isolated in a previous conventional SELEX [3]. We believe the effectiveness of the sol-gel SELEX stems from the electrostatic repulsion of aptamers by the sol-gel leaving only the highest affinity binders to interact with the sol-gel encapsulated target protein, the high ratio of target protein to other surfaces provided by the nanoporous sol-gel droplet, and the efficient and selective thermal elution of aptamers.




References

  1. "Selection and elution of aptamers using nanoporous sol-gel arrays with integrated microheaters," S.-m. Park, J. Ahn, M. Jo, D.-k. Lee, J. T. Lis, H. G. Craighead and S. Kim, Lab on a chip, 9, 1206, (2009)
  2. "Improved Sensitivity and Physical Properties of Sol−Gel Protein Chips Using Large-Scale Material Screening and Selection," S. Kim, Y. Kim, P. Kim, J. Ha, K. Kim, M. Sohn, J. Lee, J. S. Yoo, J. Lee, J.-a. Kwon, K. N. Lee, Analytical Chemistry, 78, 7392 (2006).
  3. "Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes," X. Fan, H. Shi, K. Adelman, J. T. Lis, Proceedings of National Academy of Sciences of the United States of America, 101, 6934 (2004)