devices and approaches for generating specific high affinity nucleic acid aptamers

Devices and approaches for generating specific high-affinity nucleic acid aptamers Kylan Szeto and Harold G Craighead Citation: Applied Physics Reviews 1, 031103 (2014); doi: 10.1063/1.4894851 View online: http://dx.doi.org/10.1063/1.4894851 View Table of Contents: http://scitation.aip.org/content/aip/journal/apr2/1/3?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Stable, biocompatible lipid vesicle generation by solvent extraction-based droplet microfluidics Biomicrofluidics 5, 044113 (2011); 10.1063/1.3665221 Macromolecular crowding effects on protein–protein binding affinity and specificity J Chem Phys 133, 205101 (2010); 10.1063/1.3516589 Semisynthetic DNAprotein conjugates for fabrication of nucleic acid based nanostructures AIP Conf Proc 1062, 19 (2008); 10.1063/1.3012299 Microfabricated high-performance microwave impedance biosensors for detection of aptamer-protein interactions Appl Phys Lett 87, 243902 (2005); 10.1063/1.2146058 High-resolution scanning tunneling microscopy imaging of Escherichia coli lysine transfer ribonucleic acid J Vac Sci Technol B 21, 1265 (2003); 10.1116/1.1574052 This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 APPLIED PHYSICS REVIEWS 1, 031103 (2014) APPLIED PHYSICS REVIEWS—FOCUSED REVIEW Devices and approaches for generating specific high-affinity nucleic acid aptamers Kylan Szeto and Harold G Craighead School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA (Received 28 May 2014; accepted 26 August 2014; published online 10 September 2014) High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of C 2014 Author(s) All article content, except where otherwise noted, is licensed novel aptamers V under a Creative Commons Attribution 3.0 Unported License [http://dx.doi.org/10.1063/1.4894851] TABLE OF CONTENTS INTRODUCTION CLASSICAL APTAMER SELECTIONS Basic selection principles Filtering targets: Nitrocellulose filter binding Filtering aptamers: Affinity chromatography Isolating bound complexes: Electrophoretic mobility IMPROVING CLASSICAL SELECTIONS Filtering targets: Magnetic beads Filtering aptamers: Miniaturized affinity chromatography Isolating bound complexes: Capillary electrophoretic (CE) mobility Automation and parallelization INSTRUMENTATION FOR PARTITIONING AND DIRECT READOUT OF BINDING Sorting aptamers with flow cytometry Imaging and detection with chips: Surface-bound targets 1931-9401/2014/1(3)/031103/17 3 4 4 7 Imaging and detection with chips: Surface-bound nucleic acids INTEGRATED SELECTIONS ON MICROFLUIDIC CHIPS Filtering targets: Magnetic beads Filtering aptamers: Sol-gel target immobilization Isolating bound complexes: Micro free flow electrophoresis (lFFE) Automation CONCLUSIONS AND FUTURE PROSPECTS 10 10 11 11 11 11 14 INTRODUCTION Antibodies are a class of proteins that are highly selective and have high binding affinity for foreign and potentially harmful antigens that are encountered in the body Harvested from a host organism, antibodies have become indispensable affinity reagents in research and medicine They are used for applications such as imaging specific biochemicals in cells and tissues, diagnostics to detect and quantify the presence of disease markers, and in therapeutics However, antibodies 1, 031103-1 C Author(s) 2014 V This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-2 K Szeto and H G Craighead have a number of weaknesses that limit their application For example, antibodies must be produced in vivo, which can take months and often results in problems with variability of antibody types among batches In addition, antibodies can only be generated to target molecules that elicit an immune response In contrast, a class of tight binding molecules based on nucleic acids can address the limitations of antibodies and provide new affinity reagents Polymers of nucleic acid molecules have the ability to base-pair and form complex three dimensional structures The conformational diversity that can be achieved through their unique sequences is astronomical, giving them the possibility to form structures able to recognize and bind to virtually any target molecule of interest This makes nucleic acids the perfect molecular analogues to proteins for generating functional alternatives to antibodies.1–3 These nucleic acid molecules, called aptamers, can be generated through in vitro selections This is often done through an iterative process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) SELEX involves generating a library of nucleic acid molecules (e.g., DNA or RNA) with random sequences ($1015 different sequences), screening the library for nucleic acids that bind to the target of interest, partitioning the bound molecules from the unbound molecules, and amplifying the bound molecules into a new pool enriched for good binders (Figure 1) This process can be continued until the strongest binding aptamer enriches and dominates the pool Once the enriched pool has evolved and converged to a few dozen aptamer candidates, the candidates can be sequenced, and the dominant or consensus sequences of the aptamers can be determined Although the basic combinatorial chemistry of the aptamer selection process conceptually resembles that of antibodies generated in vivo, in vitro aptamer selections provide researchers with much more freedom and control in FIG Process diagram illustrating the different in vitro selection methods The outer cycle (green) is the classical SELEX cycle which iterates through binding, partitioning, and amplifying target-bound aptamers (represented in red) The inner cycle (violet) is non-SELEX in which cycles of binding and partitioning are performed without amplification Combining both methods, RAPID-SELEX (green and violet) systematically implements non-SELEX to save time where possible but incorporates classical SELEX cycles with amplifications to replenish aptamer copy numbers or to drive up concentrations Appl Phys Rev 1, 031103 (2014) designing these affinity reagents Not only can researchers choose from an array of natural or modified nucleic acids and sequence lengths for their initial library but aptamers can be generated to bind to targets that would be toxic or non-immunogenic to an organism.4 In fact, aptamers can be generated to virtually any target from individual metal ions to whole live cells.5,6 Because aptamer selections not take place inside an organism, the environmental conditions for binding need not be physiological Instead, these conditions can be tailored to better accommodate the ionic strength/content, pH, and even temperature required for their application Most importantly, these selections can make use of the methods of molecular biology for amplifying, sequencing, and synthesizing nucleic acids This not only guarantees reproducibility between synthesis batches (the sequence is known) but also aptamers can be faster and cheaper to synthesize and modify compared to antibodies and makes sharing or acquiring new reagents as simple as reporting the sequence of the aptamer Nucleic acids are also much more stable than antibodies and can be denatured into their linear form and reversibly refolded into their active three dimensional structures, resulting in a much longer shelf life and a wider range of technological applications Lyophilized nucleic acid molecules can last almost indefinitely even at room temperature, whereas proteins and antibodies must be frozen for long term storage Finally, it is possible to link together multiple aptamers to generate multivalent aptamers that can bind multiple identical targets or bind multiple sites on a single target to enhance target recognition, or even to create aptamers that recognize novel combinations of target molecules.7–12 The potential impact for aptamers and their resultant technologies is just beginning to be understood Almost 10 years ago, the first aptamer was approved by the Food and Drug Administration to treat neovascular age-related macular degeneration.13 Since then, significant work has been done to expand the role of aptamers in imaging,14 therapeutics,15,16 targeted drug delivery,17 biosensors and integrated microfluidic, and point-of-care biomedical devices.18,19 Recently, for example, aptamers targeted to conventional therapeutic compounds have been used in integrated microfluidic chips to monitor the concentration of circulating drugs within live animals in real-time.20 This sensitive and versatile electrochemical sensor is only possible due to the conditional and reversible conformational changes that nucleic acid aptamers undergo when bound to their target molecule, as well as to their ability to be easily modified to bind to surfaces and to contain electrochemical reporters Aptamer-based microfluidic chips can be engineered to capture and purify specific targets out of complex solutions, such as cancer cells in whole blood.21 This is achieved by utilizing long surface bound DNA molecules with many linked repeats of an aptamer sequence that form highly efficient 3D affinity matrices The above technologies begin to reveal the novel capabilities of aptamers and highlight opportunities for their use in new classes of miniaturized chip-based devices However, new aptamers with enhanced functionalities are needed in order to improve their value as potent therapeutics and sensing reagents, since emerging technologies are ultimately This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-3 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) limited by the selection processes and the quality of the aptamers that are generated from them For example, higher affinity aptamers are needed to improve their sensitivity, and increased specificity is needed to reduce off-target effects such as binding to similar but undesired molecules As the demand for these powerful new molecules increases, so does the need for improved selection methods In particular, methods which incorporate miniaturized chip-based devices may be used in selections to more quickly and easily generate new aptamers with higher affinity and specificity than is currently possible These would also require only tiny amounts of target molecules, making aptamer selections available to expensive or sparsely available target molecules In this review, we discuss the range of evolving in vitro aptamer selection approaches from simple bench-top techniques to miniaturize and fully automated chip-based approaches The basic principles and contributions of each technology toward improved aptamer selections are highlighted Finally, we conclude with a discussion on the general advantages and disadvantages common among many of the technologies and propose roles that engineering and physics can play in future technology development CLASSICAL APTAMER SELECTIONS Basic selection principles The basic principles for the in vitro evolution and selection of aptamers were described over twenty years ago.1–3 This conceptually simple optimization problem requires the determination of the total concentration for both the library and target molecules that maximizes the enrichment of the highest affinity aptamer Given n molecules of different sequences, the enrichment Ei for a molecule of type i can be expressed as the ratio of its fractional representation among all bound molecules to its starting fraction P ẵT : Si = niẳ1 ẵT : Si ; (1) Ei ẳ ẵT : Si ỵ ẵSi ị=ẵST where [T:Si] and [Si] are the concentration of bound and unbound molecules of type i, respectively This must be maximized for sequences with the highest affinity (lowest dissociation constant Kd) given the set of n equilibrium binding equations ẵT : Si ẳ ẵSi ẵT i ẳ 1n; Kd;i ỵ ẵT (2) where [T] is the unbound target concentration and Kd,i is the dissociation constant for molecules of type i Using the conservation equations ½TŠT ẳ ẵT ỵ n X ẵT : Si ; (3) iẳ1 ẵST ẳ ẵS ỵ n X ẵT : Si Š; (4) i¼1 where [S] is the concentration of all unbound molecules, the optimum total concentrations for the target [T]T and the library [S]T that maximize Eq (1) for high affinity sequences can be determined This problem cannot be solved analytically for n > and must be solved separately for each selection cycle as the relative frequency of sequences changes after every binding step, thus minimizing the total number of selection cycles needed to converge the pool to a single aptamer sequence A number of theoretical efforts have attempted to address this optimization problem using numerical methods and/or approximations.22–35 These theories assume equilibrium solution binding, because time dependent models double the number of parameters (Kd is the ratio of the kinetic binding off-rate and on-rate: koff/kon) and generally require a significant amount of prior information about the initial library, such as the distribution of sequences ([Si]) and affinities (Kd,i) to the target In contrast, many selections are not performed in true equilibrium or with free molecules in solution, and little to no information is known about the initial library or its interaction with the target In addition, the molecules cannot always be considered point particles as selections may be affected by molecule size and orientation.36,37 Although some theoretical work has been done to fill these information gaps,38–42 most parameters remain experimentally unknown, and researchers have adopted simple intuitive schemes to attempt to create competitiveness and stringency during selections, such as gradually increasing the ratio of library to target molecules (i.e., the fold-excess of library to target) and/or reducing the quantity or concentration of target molecules These selections are typically performed in one of three ways (1) Filtering target molecules out of solution and thus retrieving bound aptamers (2) Filtering aptamers out of solution through a stationary phase of immobilized target molecules (3) Spatially resolving and isolating targetaptamer complexes from unbound molecules by electrophoretic mobility differences Filtering targets: Nitrocellulose filter binding One of the first methods of selection was nitrocellulose membrane filtration, which corresponds most closely to the theoretical models mentioned above (Figure 2(a)) Generally, the target molecules and the nucleic acid library are mixed together in solution and allowed to approach equilibrium Target molecules and aptamers bound to them can then be partitioned from the solution by rapidly filtering the mixture over a nitrocellulose membrane.3,43–45 The membrane concentrates target-bound nucleic acids and allows unbound nucleic acids to pass through This process is fast and straight forward, and it is one of the most common partitioning methods used for selecting aptamers However, because the non-specific affinity of nitrocellulose to amino acids is central to this technique, it only works well with protein targets In addition, the large surface area of the filter enables a significant amount of free nucleic acids to nonspecifically adsorb to the membrane This nonspecific binding can significantly hinder the enrichment of target-binding aptamers or completely dominate the enriched pool Extensive washing, or negative selections that remove these filter-binding molecules, could be used to improve aptamer enrichments.46 This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-4 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) FIG Schematics for the classical technologies used to partition aptamers (red) from non-binding nucleic acids (blue) when selected against target molecules (orange) (a) Membrane filtration is used to non-specifically capture target molecules along with any bound aptamers from an equilibrium mixture Unbound nucleic acids pass freely through the filter (b) Affinity chromatography utilizes a column of packed resin which is functionalized with target molecules, and a library or pool of nucleic acids which is passed through the column Aptamers are captured on target molecules, while unbound nucleic acids pass freely through the column (c) EMSA use gels to separate equilibrium mixtures containing bound and unbound nucleic acids and targets By applying an electric field E, bound and unbound molecules, which have different sizes and charges, will migrate at different rates (given by l1 and l2) through the gel, which prevents the isolated populations from mixing Filtering aptamers: Affinity chromatography Another method that was initially applied to aptamer selections is affinity chromatography (Figure 2(b)) This technology is traditionally used to separate and purify components from a mixture of molecules, some of which have a specific affinity to or interaction with a stationary phase (resin packed into a column) through which the mixture flows By immobilizing target molecules to an affinity resin, the injected nucleic acid library becomes enriched for targetbinding aptamers, which are retained within the column Non-binding nucleic acids simply flow through as waste Aptamer-bound targets can then be chemically eluted off of the resin In contrast to nitrocellulose membrane filtration, affinity chromatography can be used to immobilize proteins as well as small molecule targets.1 Given its simplicity and familiarity among many laboratories, affinity chromatography has become the dominant method for small molecule selections.46–50 However, this method generally requires large quantities of target to achieve sufficiently high loading onto the entire column and can suffer from non-specific binding of the nucleic acids to the resins, requiring extensive washing or negative selections.46 In addition, this method requires the incorporation of an affinity tag to target molecules for immobilization; and although this is commonplace for proteins, it can be difficult to achieve for small molecules and restricts the modes for aptamer binding Isolating bound complexes: Electrophoretic mobility Early selection methods also included Electrophoretic Mobility Shift Assays (EMSA) (Figure 2(c)) Like the older nitrocellulose filtering method, these selections allow targetlibrary mixtures to equilibrate together in solution However, here the bound and unbound populations can be spatially separated by adding the mixture to a gel and applying an electric field E.51,52 Depending on their shape, size, and charge, each population has a different mobility le that causes unbound target, unbound nucleic acids, and bound complexes to migrate through the gel with different velocities v v ¼ le E: (5) The gel prevents mixing or significant dispersion of the isolated populations that would otherwise take place in solution The band containing bound complexes can then be imaged with radioactivity (or fluorescence), cut out, and crushed to allow the aptamers to easily diffuse back into solution This selection method almost completely eliminates background binding as well as the need for washing or negative selections However, the modifications required, especially radioactivity, are generally undesirable, and selections with different targets can have differing results and make separations difficult to resolve, especially with small targets In addition, the separation step is slow and can be far from equilibrium, providing opportunities for bound complexes to dissociate during the lengthy process IMPROVING CLASSICAL SELECTIONS Filtering targets: Magnetic beads One of the advantages of filter binding is its ability to easily and rapidly concentrate aptamer-bound target molecules, while separating them from a solution of unbound nucleic acids However, due to the nature of the filters, these selections suffer from background binding and are limited to proteins To address some of these limitations, techniques utilizing magnetic beads were devised (Figure 3(a)).53,54 Magnetic bead-based SELEX allows selections to be performed to any target that can be immobilized onto the beads In addition, selections can be performed in smaller volumes with much less target, and the bead-bound target can be rapidly concentrated and isolated from the bulk solution simply by using a permanent magnet The magnetic beads can then be aggressively washed and concentrated again if needed and used directly in Polymerase Chain Reaction (PCR) amplifications This has allowed aptamers to be selected in a single cycle By repeatedly washing away unbound/dissociated nucleic acids and re-equilibrating the remaining bead-bound aptamers, only the strongest binding aptamers remain in the This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-5 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) FIG Schematics for improved variations on the classical technologies used to partition aptamers (red) from non-binding nucleic acids (blue) when selected against target molecules (orange) (a) Magnetic beads are used to capture target molecules along with any bound aptamers from an equilibrium mixture into a small localized pellet Unbound nucleic acids remain in the bulk solution (b) Capillary affinity chromatography utilizes a capillary with its inner wall functionalized with target molecules, and a library or pool of nucleic acids which is passed through the capillary Aptamers are captured on target molecules, while unbound nucleic acids pass freely through the capillary This flow regime can achieve such high resolutions that individual binding species (i, j, and k) can be resolved into separate bands and isolated (c) Capillary electrophoresis uses the high resolution and non-mixing regime of capillaries to separate equilibrium mixtures containing bound and unbound nucleic acids and targets By applying an electric field E, bound and unbound molecules, which have different sizes and charges, will migrate at different rates (given by l1 and l2) through the capillary, which prevents the isolated populations from mixing end.55 A variation on this technology uses magnetic beads in a slightly different manner In a method called Aptamer Selection Express (ASExp), target molecules and the library are mixed together in solution just like in filter binding or EMSA.56 However, in this case, the library consists of double stranded nucleic acids that must dissociate in order to reveal a single stranded aptamer This imposes a binding threshold on potential aptamers and allows distinguishing single stranded target-binding aptamers from double stranded nonbinding nucleic acids This difference is exploited by recovering the single stranded aptamers using magnetic beads coated with long random sequences of single-stranded nucleic acids, allowing aptamers to be selected in a single cycle using this technique In another variation of this technique, the library is fluorescently labeled in order to quantify the level of binding after each cycle of SELEX This method, called FluMagSELEX, allows sensitive measurements of binding to be made due to the efficient concentration of target-bound nucleic acids on the magnetic beads and eliminates the need for radioactivity-based measurements.57 Magnetic bead-based techniques are of interest because the ability to manipulate the beads using magnetic fields creates the potential for more sophisticated and automated technologies A recent example of this, called Magnetic-Assisted Rapid Aptamer Selection (MARAS), utilizes a magnetic field to not only isolate bead-bound aptamers but also actively remove more weakly bound aptamers.58,59 This additional discrimination is achieved by placing an equilibrated mixture within a solenoid and applying an alternating current This results in an alternating magnetic field, the strength and frequency of which can be adjusted such that lower affinity aptamers begin to dissociate from the target molecules This is due to the viscous dissipative forces imparted to the bound aptamers as the beads oscillate within the field, enabling selections to be performed in only a single cycle This approach has been shown to generate aptamers for which the affinity increases with increasing magnetic field strength and frequency Filtering aptamers: Miniaturized affinity chromatography There have been several new technology developments involving affinity chromatography One example has been the miniaturization of affinity chromatography through the use of microcolumns, which are orders of magnitude smaller than conventional columns By slowly injecting nucleic acids into the packed microcolumns, the entire library is efficiently sampled and high affinity aptamers are retained within the small column The use of microcolumns was optimized for maximum enrichments of aptamers, revealing critical target concentrations that could be explained by geometric constraints for steric hindrance.60,61 This concentration as well as the small column volume reduce the amount of target needed by several orders of magnitude Furthermore, tests that varied the flow rates resulted in enrichment trends that scaled oppositely from the time-dependent kinetic binding model where the concentrations of unbound and bound molecules are also dependent upon their position x along the column dẵT : Si xị ẳ kon;i ½T ðxފ½Si ðxފ À koff ;i ½T : Si ŠðxÞ: dt (6) This allows rate dependent discrimination between aptamers via shear forces and other mechanisms analogous to the MARAS method described above Also using the microcolumns, a modified SELEX method, called RNA APtamer Isolation via Dual cycles or RAPID-SELEX was demonstrated that generalized the SELEX method to permit the systematic skipping of amplification steps (Figure 1).62,63 This is This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-6 K Szeto and H G Craighead advantageous as it significantly reduces the time required for selections by allowing multiple cycles of binding to be performed in effectively fewer “rounds” of SELEX However, despite producing improved aptamer enrichments, RAPIDSELEX is viewed traditionally as less competitive than conventional SELEX due to diminishing nucleic acid concentrations between infrequent amplifications Together, these results highlight the importance of empirical characterization and optimization of selection technologies Interestingly, multiple microcolumns can be connected together allowing for simultaneous in-line negative selections, multiplexing, and parallelization Recently, this technology was scaled up to a Microplate-based Enrichment Device Used for the Selection of Aptamers (MEDUSA) and has a 96-well microplate format to allow high-throughput and potentially automated platebased selections and processing to be performed.61 In a similar modification of affinity chromatography, MonoLEX utilizes a narrow affinity column in the form of a capillary.64 As is typical of chromatographic separations, the nucleic acid library can resolve itself along the capillary into different populations of binders (Figure 3(b)) In a manner analogous to connecting and disconnecting microcolumns in series, by physically cutting the capillary column into small fragments (i.e., $40 segments), aptamer populations can be isolated and recovered from each fragment individually and characterized, with the highest affinity aptamers generally residing in earlier segments The capillary columns’ efficient separation of subpopulations into narrow and well-resolved distributions allows MonoLEX to isolate aptamers in a single cycle Affinity chromatography has also been taken to the limit of miniaturization by performing SELEX to target immobilized on a single bead.65 With this method, aptamers are generated by incubating a single target-functionalized microbead with a fluorescently labeled nucleic acid library By significantly reducing the number of target molecules, it is assumed that only the highest affinity aptamers can be bound, due to competition with low affinity and non-binding nucleic acids for the few available binding sites This can be seen in the limit where the concentration of unbound target [T] approaches zero " #À1 n X ½T Š½SŠ ðKd;i ị1 ẵSi : (7) % hKd i ẳ ẵST ½T : SŠ i¼1 [T:S] is the total concentration of bound aptamers (and target), so as the concentrations of unbound nucleic acids [Si] go up, the average of the dissociation constant hKdi of all bound aptamers decreases Once washed, the bead can be collected and subjected to PCR amplification The fluorescently labeled library used in this method allows the level of binding to the bead to be monitored under a microscope during/after each cycle, and the bulk binding affinity to target molecules to be determined quickly via fluorescence anisotropy Using this technique, high affinity aptamers were generated in just two cycle of SELEX, but only a single cycle may be necessary Although not demonstrated, this simple process could be scaled up to include multiple single-target beads or even automated However, identifying, isolating, Appl Phys Rev 1, 031103 (2014) and handling single beads may limit this technology’s capacity for more streamlined processing Isolating bound complexes: Capillary electrophoretic (CE) mobility As discussed above, target-binding aptamers can be distinguished from non-binding nucleic acids by a change in mobility when bound to target molecules This is achieved primarily due to the differences in the net charge between bound and unbound molecules EMSA-SELEX has the advantage of equilibrium binding and (non-equilibrium) separations on a gel, which eliminates the need for washing and negative selections However, the use of radioactivity and the need to cut and process gels makes this method tedious and difficult to adapt for higher throughput or more automated selections This limitation has been overcome through the application of CE CE-SELEX utilizes integrated fluidics with an electric field applied across the system (Figure 3(c)) Using a capillary with a small internal diameter allows separations to occur in solution rather than requiring a gel (see Eq (5)).66–70 This is made possible by the laminar flow regime (low Reynolds number) in the capillary, which is largely free of turbulent flow and mixing (other than diffusion) UV detectors can be used to identify the band of bound complexes as populations of molecules migrate through the capillary, eliminating the need for radioactivity These selections are fast and work well with large target molecules and result in efficient and high resolution separations However, selections using different targets can vary greatly, and the optimal binding and running conditions may need to be determined beforehand In addition, separations with targets that only result in modest shifts in mobility can be nearly impossible to resolve Resolution can be particularly problematic if the capillary is overloaded with nucleic acids that can obscure the desired aptamer band Therefore, CESELEX can only handle small volumes ($100 nl) and use starting libraries at incredibly high concentrations and several orders of magnitude less diverse (1012–1013) than conventional selections in order to prevent overloading and to achieve good separations With appropriate modifications, CE-SELEX has been used to estimate binding affinity during selections This is because information can be obtained about the populations of unbound target, unbound nucleic acids, as well as the bound complexes and their gradual dissociation as they migrate past the detector.71 In addition, distinguishing the various populations can be made easier by incorporating fluorescence capabilities into the selection A novel CESELEX method based on these modifications, called NonSELEX, was demonstrated, which also completely eliminates all amplification steps allowing aptamers to be generated in a single “round” comprising multiple binding cycles (Figure 1).72 This method involves collecting the band of bound aptamers and re-injecting them into the CE system for additional cycles of purification However, due to the small volume constraints, only a tiny fraction of the collected pool can be re-injected for the next cycle, significantly limiting the number of nucleic acids that can be sampled Some This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-7 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) recent optimization has been done using larger capillaries and multiple pool injections to improve sampling after each cycle.73 Automation and parallelization In addition to develop new and more efficient selection methods, researches have also accelerated aptamer discovery through robotic technologies For example, semi-automated and parallel selections are possible using target-functionalized magnetic beads and microplates Using an array of magnetic wands, magnetic beads and their bound aptamers can be captured, removed from solution, and transferred to fresh wells on the microplate, allowing the automation of most of the selection steps apart from amplifications.74,75 Completely automated selections have also been demonstrated that use magnets to retain beads in the microplate wells as solutions are exchanged instead,76 although these protocols were later changed to incorporate membrane filtration to capture and wash the magnetic beads.77–80 Affinity columns have also been used in automated selections to filter out affinity-tagged targets and bound aptamers.81 However, an example that does not require any filtering uses simple immobilization of targets directly to the surfaces of microplates.82 As an alternative to generally low-throughput automation, substantial scaling up though massive parallelization of selections allows the average time per target to be reduced proportionally, assuming steps for each target can be performed simultaneously Using a similar target-functionalized 96-well microplate, multiplexed and massively parallelized SELEX has been demonstrated through simultaneous processing and analysis.83 In addition, the 96-well microplatebased affinity microcolumns discussed earlier were also used to perform simultaneous processing and analysis.61 These highly parallelized technologies are ideal compliments to automate microplate-based protocols and combining these two approaches may allow researchers to achieve automated and massively parallelized selections using similar robotic systems INSTRUMENTATION FOR PARTITIONING AND DIRECT READOUT OF BINDING Sorting aptamers with flow cytometry As interest in aptamers has grown, researchers with expertise outside of classical SELEX have begun to recruit sophisticated systems and instruments to help perform selections This has been particularly fruitful with techniques that not only separate populations but also provide information about the bulk binding behavior of the aptamer pools, as with CE-SELEX One of these methods uses flow cytometry, which is usually used to count or measure properties of cells at very high rates by rapidly interrogating individual cells in a flow stream (thousands per second) A specialized form of flow cytometry, called Fluorescence-Activated Cell Sorting (FACS), can be used to separate different populations This is done by breaking up the flow stream into individual droplets and placing a charge on each droplet depending on its contents’ fluorescence characteristics (Figure 4(a)) As the FIG Schematic for bead-based selections utilizing FACS (a) Beads, each of which is coated with many copies of nucleic acid molecules of the same sequence, are partitioned into aptamers (red) and non-binding nucleic acids (blue) based on binding to fluorescently labeled target molecules (orange) Fluorescence measurements of each bead are used to identify brightly labeled beads which contain tight binding aptamers and are sorted by imparting a charge on each bead and deflecting them with an electric field (b) An example of a brightly labeled bead indicating the presence of a tight binding aptamer candidate Reproduced by permission from Yang et al., Nucleic Acids Res 31(10), 54e (2003) Copyright 2003 Oxford University Press droplets fall, they are electrostatically deflected and separated into collection tubes according to their charge These systems have been applied to aptamer selections against whole cells using a method called FACS-SELEX, which is useful not only for quantifying and sorting aptamer-bound This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-8 K Szeto and H G Craighead cells from the bulk solution of unbound nucleic acids but can also be used to separate specific populations of cells, such as living cells from dead cells.84,85 A key advantage of flow cytometric systems is the ability to probe one cell at a time Therefore, a natural modification of these systems involves replacing cells with beads Selections with beads that are each bound to only a single type of nucleic acid sequence have been demonstrated where high affinity beads are identified by incubating them with their target and imaging the level of binding with fluorescently labeled targets86 or antibodies (Figure 4(b)).87 This method is unique in that the highest affinity aptamers are assayed and identified directly by the brightest beads These beads can be picked up via a micropipette and sequenced, allowing selections to be completed in a single cycle In a method called Monoclonal Surface Display SELEX (MSDSELEX), individual nucleic acid sequences enriched from traditional pre-selections can be rapidly screened in a single cycle.88 This can also be achieved using agarose beads which contain the clonal sequences internally rather than on their surface.89 Using multiple colors, additional or alternative binding requirements (which are assayed fluorescently) can be imposed A simple two-component test mixture of beads was used to demonstrate this kind of multicolor FACS detection Recently, a true single sequence/bead selection called particle display utilizing the sorting capabilities of FACS was fully demonstrated.90 Currently, FACS technologies are restricted to detect and sort about 108 total beads/cells which significantly reduces the number of nucleic acids that can be screened in this format Particle display resolves this sampling limitation by performing the first selection cycle without FACS so that a full size library can be screened, generating a partially enriched pool which can be much more efficiency utilized via FACS Interestingly, the fluorescent signature of target binding to the beads not only allows a proportional readout of binding affinity but the near bulk binding characteristics of $105 copies of each nucleic acid sequence on its bead also overcomes the stochastic binding of single complexes and allows for unparalleled sensitivity and confidence in discriminating and sorting between high and low affinity aptamers Currently, FACS systems can also separate mixtures into six subpopulations or distribute individual sorted objects into the wells of microplates This highlights FACS-based methods’ scalability, where the ability to analyze and sort using multiple colors and sorting channels can be advantageous for multiplexing or high-throughput parallelization through downstream microplate processes, especially for selections directly to fluorescent targets such as fluorescent proteins or dyes.91 Imaging and detection with chips: Surface-bound targets The ability to directly observe and image interactions between aptamers and target molecules has advantages over a typically blind selection strategy This can be as simple as observing binding events under an optical microscope, such as with the FACS-based methods However, it is often difficult to fluorescently label target molecules, and this can have Appl Phys Rev 1, 031103 (2014) undesirable consequences in aptamer-target recognition Furthermore, adding fluorescent antibodies complicates the selection process and is not always a possible alternative A more general and straight forward approach involves fluorescently labeling the nucleic acid library, as in the single-bead affinity selection discussed earlier In addition, imaging can be done easily by immobilizing target molecules on a flat substrate Selections can be done by incubating a targetfunctionalized coverslip with a fluorescently labeled library The coverslip can then be extensively washed and imaged under a microscope to find bright and highly localized spots indicating aptamer binding, and the aptamers recovered thermally through heat elution in solution Using this simple method, an inexpensive and rapid one-step (single cycle) selection was demonstrated.92 Simply monitoring selections in this way is useful to ensure that binding is taking place and to assess the degree of background binding, which may require additional washing or negative selections In addition, the use of fluorescently labeled target molecules with the fluorescent library can be used to image both simultaneously and find co-localized spots of aptamers binding specifically to target molecules on the surface This ability to visually discriminate between target-aptamer interactions from non-specific background binding can have significant advantages for improving aptamer enrichments A similar selection method, called NanoSelection, utilizes an Atomic Force Microscope (AFM).93 This method incorporates fluorescently labeled nucleic acids attached to beads using a similar single sequence/bead scheme as the FACS-based methods Using a fluorescence microscope-AFM hybrid system, beads that bind to a target-functionalized chip are imaged using the fluorescence microscope The AFM is then used to generate a local image of the bead on the chip, and then the AFM’s tip is used to physically “spear” or displace the bead from the surface for retrieval and analysis NanoSelection was demonstrated using a two-component test mixture of aptamers with non-aptamers but is best suited to small libraries on beads In addition to locating bound beads, it should be possible to observe their dynamics while in solution, using their bound-state fluorescence intensity and duration to pinpoint the most tightly bound beads as well as their mobility when unbound, to determine relative binding affinities between candidate beads In another method called AFM-SELEX, the library is biotinylated and bound directly to a streptavidin-functionalized AFM tip instead of to beads (Figure 5(a)).94 This method is particularly interesting because as the tip probes the target functionalized chip, nucleic acids in the library that have an affinity for the target are forced to compete against the biotinstreptavidin interaction This interaction is quite strong and acts as a binding threshold that aptamers must surpass in order to detach from the tip and remain bound to the chip In doing so, images of the chip surface as well as thousands of force curves are generated (Figure 5(b)) Successful selections using AFM-SELEX showed gradual increases in the average force exerted on the AFM tip using adhesion force analysis during each selection cycle These results clearly indicate the enrichment of higher affinity aptamers to the target Although this method provides a competitive binding This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-9 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) FIG Schematic of three chip-based technologies used to partition aptamers (red) from non-binding nucleic acids (blue) when selected against target molecules (orange) (a) AFM utilizes a library or pool of nucleic acids bound to the AFM’s probe tip By running the tip over a surface of target molecules which are immobilized onto a chip, tight binding aptamers which can overcome their binding energy to the AFM tip can detach and bind to target molecules on the surface (b) Example force and height AFM images acquired using a nucleic acid-coated probe tip on a target coated chip’s surface Reproduced by permission from Miyachi et al., Nucleic Acids Res 38(4), e21 (2010) Copyright 2010 Oxford University Press (c) SPR utilizes target molecules which are immobilized onto a chip By flowing a library or pool of nucleic acids over the chip, tight binding aptamers bind to target molecules on the surface while non-binding nucleic acids flow past This can be imaged and quantified through proportional changes in the surface plasmon conditions The dielectric constants e determine the plasmon angle hK for an incident laser of frequency x (d) Example SPR response curves for various enriched pools showing the binding and unbinding of enriched aptamer pools Reprinted with permission from T S Misono and P K R Kumar, Anal Biochem 342(10), 312–317 (2005) Copyright 2005 Elsevier (e) Microarrays utilize a library of predetermined nucleic acid sequences which are then addressed and synthesized in situ on specific elements of the chip array By exposing fluorescently labeled target molecules to the array, tight binding aptamers bind to the target molecules allowing each aptamer to be individually identified through fluorescence imaging (f) Example fluorescence image of individual nucleic acid sequences on a microarray binding florescent targets Reproduced by permission from Fischer et al., PloS One 3(7), e2720 (2008) Copyright 2008 Fischer et al threshold and data regarding the bulk binding behavior of the library and enriched pools, this technique has significant limitations in the number of nucleic acid molecules that can be bound to the AFM tip and probed and is not easily scalable for higher throughput aptamer selections A technique that is naturally sensitive to surface bound molecules is Surface Plasmon Resonance (SPR), which excites plasmons in thin metal films and uses their sensitivity to the refractive index of material near their surface to monitor binding kinetics (Figure 5(c)) This is typically achieved by reflecting p-polarized laser light of frequency x, in a medium with dielectric constant of e3 , off of a thin film of metal (i.e., gold) and monitoring changes in the reflectance minimum at angle hK that satisfies the plasmon resonance condition for wavenumber KðxÞ As molecules bind to and unbind from the surface, they change the effective dielectric constant e1 of the medium in contact with the metal of dielectric constant e2 x K x ị ẳ c r e1 e2 x pffiffiffiffi e3 sin hK : ¼ c e1ỵ e2 (8) This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-10 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) One key advantage of SPR systems is that they integrate fluidics and flow cells with surface-bound target selections and can have several flow channels, which potentially allow for multiplex or parallelized aptamer selections By injecting and exposing nucleic acids with concentration [S] to the chip surface, the pool’s bulk on-rate kon for binding can be estimated as Rtị ẳ RMAX ẵS ekon ẵSỵKd ịt ; Kdỵ ẵS (9) where the R(t) is the time-dependent response of the SPR resonance angle and RMAX is the maximum signal Since this measurement is performed under flow, the concentration of unbound nucleic acids is constant, making the binding trends independent of the target concentration In addition, washing/elution of aptamers off the chip allows the bulk off-rate koff for unbinding to be estimated as they slowly dissociate and are collected for future selection cycles RðtÞ ¼ Rð0ÞeÀkof f t : (10) SPR in SELEX was initially demonstrated as a clean-up selection following many cycles of nitrocellulose filter binding and was used to determine the bulk binding affinity of the final pool.95 However, SPR has also been utilized as the sole SELEX platform showing steady increases in the timedependent binding rates after each cycle and indicating the enrichment of higher affinity aptamers (Figure 5(d)).96–98 In some cases, multiple fractions were collected during the dissociation step Although not demonstrated, the highest affinity aptamers are expected to take longer (on average) to dissociate due to a smaller koff and hence reside primarily in the later fractions Fractionating the dissociation phase could be used as an off-rate threshold for aptamers in SPRSELEX However, although off-rates can be determined easily in SPR, accurate determination of the on-rate and the equilibrium binding affinity requires multiple concentrations of the library/pool to be probed in order to fit all the parameters of Eq (9) In addition, since the determination of the onrate requires the concentration of unbound nucleic acids to remain constant at all times, limits on the possible flow rates that can be used have to be imposed, which must be low enough for efficient sampling but high enough to overcome mass limited transport Imaging and detection with chips: Surface-bound nucleic acids Performing selections with surface-bound molecules has resulted in a range of varying chip-based methods With the emergence of technologies such as microarrays, precise localization and addressing of many different molecules onto a single chip can be achieved This has been applied to multiplex and parallelize chip-based selections with multiple surface-bound target molecules and concentrations.99–102 Although initially only a few dozen isolated spots were placed manually on chip surfaces, automated and robotic systems have enabled much higher density arrays of molecules to be generated Using modern lithographic techniques for microscale patterning, microarrays can now be fabricated with nearly 105 individual spots This high-density configuration has been adopted as a means to probe individual sequences of a nucleic acid library with sensitivities and sampling depths comparable to particle display using FACS (Figure 5(e)) For aptamer selections, random and unique nucleic acid sequences are individually synthesized in situ and addressed on predetermined spots on the microarray Fluorescently labeled target molecules can then be exposed to the array and imaged to identify potential aptamers via bright localized spots (Figure 5(f)) In a selection method called CLADE (Closed Loop Aptameric Directed Evolution), a set of the brightest spots ($1%) are identified and their associated sequences are controllably mutated and synthesized onto a new chip for additional selection cycles.103,104 This is comparable to performing a pre-selection to compensate for the lower sampling capabilities of the microarray By mutating the top performing sequences, tighter binding variants can be identified and further matured Although microarray-based selections such as CLADE have great potential, they require a small starting library of individually synthesized nucleic acids with predetermined sequences For this reason, microarrays have also been used post-selection to screen enriched pools which contain significantly fewer unique sequences and even fewer aptamer candidates, which can be identified through sequencing and chosen based on population metrics such as multiplicity or enrichment.105–107 Aptamers isolated from selections can also be studied and optimized through mutations and assayed on microarrays to identify key features which are critical to the highest affinity aptamer such as the minimal aptamer structure, structure-function relationships, as well as conserved sequence motifs.107–109 The majority of these selection microarrays rely on the imaging and detection of fluorescently labeled molecules (target or library) However, since microarrays share a chip format with a number of label-free detection methods, arrays of nucleic acids have been generated and screened without fluorescent modifications made to them or the target Using an array of electrodes, nucleic acids on the array which are bound to gold nanoparticles can generate measurable current and/or voltage signals when bound to target molecules via electrochemical detection.110 Although this technique can be used to screen multiple aptamers simultaneously and is label-free, this method can only work with target molecules that are electroactive and will generate a sufficiently large signal upon binding to the electrode As an alternative, arrays of aptamers have also been generated and screened using SPR, which does not require fluorescent labels or special properties from the target molecules.111 INTEGRATED SELECTIONS ON MICROFLUIDIC CHIPS Researchers are interested in miniaturizing selection technologies onto chips, as well as their automation Initial efforts aimed to miniaturize conventionally large robotic systems used in automated SELEX.112 This was done through the integration of a fluidic microchip and was used to successfully select an aptamer through an automated process This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-11 K Szeto and H G Craighead that incorporates all the steps from binding to amplification Although the binding step takes place off-chip in an affinity capillary and valving and other fluidic controls are performed using large external fluidic instruments, this microfluidic SELEX (MSELEX) platform is a significantly smaller and simpler system than other automated technologies and represents an important step toward more integrated chip-based solutions for fluidic selections Recently, significant research efforts have focused on capitalizing even more on the micro- and nanoscale processes used in the fabrication of functional chips This has led to the development of a number of devices that can be used to integrate and perform a number of selections steps These new selections benefit not only from the reduced volumes but also from the scalability of the fabrication methods, putting chip-based multiplexed, and automated aptamer selections within reach Filtering targets: Magnetic beads Due to their easy manipulation, magnetic beads have been incorporated into microfluidic systems to further improve the efficiency of aptamer selections One of the first such devices is described in a method called M-SELEX (Figure 6(a)) Here, a library is incubated with target-functionalized magnetic beads and injected into a Continuous-flow Magnetic Activated Chip-based Separation (CMACS) device.113 Magnetic beads and bound aptamers are then magnetophoresed out of laminar input streams, into a non-mixing center stream for collection This is done through a magnetic field gradient generated by integrated ferromagnetic structures Together, the magnetic beads and the CMACS device allow highly stringent selections with very low concentrations of bead-bound targets to be efficiently performed (see Eq (7)) and have resulted in an aptamer being isolated in a single cycle A simpler MicroMagnetic Separation (MMS) chip was also demonstrated that does not limit the magnetic beads’ residence time in the microfluidic chip In this case, ferromagnetic grids are used to capture and retain magnetic beads as they flow into the device, allowing a wider range of flow rates, as well as long and extensive washes to be performed in the channel.114,115 In a technique utilizing dilutions116 called Volume Dilution Challenge (VDC), VDC-MSELEX uses the MMS chip to capture and re-concentrate magnetic beads following dilutions of up to one thousandth of the equilibrated solution, which helps to irreversibly dissociate aptamers with poorer off-rates.117 M-SELEX has also been coupled to highthroughput sequencing (HiTS) in a method called Quantitative Selection of Aptamers through Sequencing (QSAS), to analyze the copy numbers and enrichments of millions of aptamer candidates in multiple pools.118 Recently, this has been further improved through a Quantitative Parallel Aptamer Selection System (QPASS), which utilizes a microarray of thousands of the top multiplicity aptamer candidates to measure each of their binding affinities in parallel.106 Filtering aptamers: Sol-gel target immobilization A chip-based technology with similar characteristics of affinity chromatography was developed using sol-gel arrays as a solid support for target immobilization (Figure Appl Phys Rev 1, 031103 (2014) 6(b)).119–121 Selections are achieved by trapping target molecules within a small drop of highly porous 3D silica matrix Nucleic acids are gently flowed across a sol-gel spot allowing them to enter and diffuse within the sol-gel and bind to the target in its native state A microfluidic chip is used so that the injections of the library are confined close to the solgel spots thereby increasing the efficiency of sampling This chip also contains integrated microheaters, which allow bound aptamers to be thermally eluted Serialized multiplexed selections using this sol-gel device were demonstrated Recently, valving was integrated into the device to allow changes to the fluidic network such that the parallelized multiplex sol-gel selections can occur while minimizing cross-contamination between spots during elution.122,123 These integrated multiplex selections along with the significantly reduced amount of target are very attractive; however, the binding and elution of sequences in the sol-gel spots are diffusion limited and may reduce the number of nucleic acids that can be effectively sampled A more conventional chromatographic device was recently demonstrated that uses pairs of on-chip microcolumns which overcome these sampling limitations and allow for multiplexing and simultaneous negative selections.124 Isolating bound complexes: Micro free flow electrophoresis (lFFE) Mobility separations have also been integrated into microfluidic chips, taking advantage of the highly efficient selections that can be achieved by CE-SELEX By integrating electrodes, bound and unbound nucleic acids are separated by their different mobilities (Eq (5)) However, in normal CESELEX, the amount of library that can be injected is significantly limited by the separation resolution of the capillary This restriction can be eliminated by using lFFE, which utilizes a lateral electric field to continuously separate large volumes of equilibrated sample sideways as its being injected forward under hydrostatic pressure (Figure 6(c)).125 Furthermore, the output of the device is partitioned into two streams allowing fluorescently labeled bound and unbound nucleic acid populations to be imaged and continuously directed into separate collection fractions by adjusting the fluid flow rate and the electric field strength lFFE also eliminates the need to time the collection of desired fractions as in CE and was used to successfully isolate an aptamer in only a single cycle from a library over 100-fold larger than CE Automation A key advantage for chip-base selections is the ability to fully integrate the entire selection process into a single device and ultimately automate it Towards this goal, selection devices have been designed to incorporate advantages from various technologies into a single microchip For example, one such microfluidic device has two chambers connected by a channel filled with gel (Figure 7(a)).126–128 One chamber contains integrated microheaters and is packed with target molecules immobilized onto microbeads forming a microcolumn Fluorescently labeled nucleic acids are flowed through the microcolumn and captured onto the target- This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-12 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) FIG Examples of three microfluidic devices that integrate the basic methods of partitioning aptamers from non-binding nucleic acids (a) A microfluidic device which can capture and partition target-functionalized magnetic beads out of a solution equilibrated with a library or pool of nucleic acids Microfabricated ferromagnetic structures capture and direct magnetic particles as they are injected into the device from the outer two left inlets, diverted (region I), and isolated into a central collection stream (region II) free from unbound nucleic acids which are carried away in the outer two waste outlets (region III) on the right Reproduced by permission from Lou et al., Proc Natl Acad Sci U S A 106(9), 2989–2994 (2009) Copyright 2009 National Academy of Sciences (b) A microfluidic device which utilizes a silica sol-gel matrix to immobilize and isolate multiple target molecules in a single channel A library or pool of nucleic acids is passed (from left to right) over the target sol-gel spots S1 through S5, allowing aptamers to diffuse and bind to each target within the sol-gel spots Unbound nucleic acids pass freely out of the device, and then aptamers are collected from each sol-gel spot using individual electrodes to heat and dissociate target-bound aptamers allowing them to diffuse out of the sol-gel back into solution Reproduced by permission from Park et al., Lab Chip 9(9), 1206–1212 (2009) Copyright 2009 The Royal Society of Chemistry (c) A microfluidic device which integrates electrodes to achieve lateral electrophoresis The left is a diagram of the device which uses a buffer stream (inlet at 1) to separate a continuously injected equilibrium mixture (inlet at 2) Bound and unbound molecules are separated orthogonally (Green lines) to the bulk flow and isolated into different collection channels (outlets at 3) by using the electrodes (4) to exploit differences in their electrophoretic mobility The top-right panel shows a fluorescence image of the unbound (1) and bound (2) molecules just before entering the two collection channels Bottom-right is a corresponding intensity plot of the two populations The arrow indicates the split point for the two channels Reproduced by permission from M Jing and M T Bowser, Lab Chip 11(21), 3703–3709 (2011) Copyright 2011 The Royal Society of Chemistry This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-13 K Szeto and H G Craighead Appl Phys Rev 1, 031103 (2014) FIG Examples of three fully integrated microfluidic devices which can perform and automate all of the selection steps (a) A microfluidic device which uses target immobilized onto beads (left Blue chamber) to capture injected nucleic acids Bound aptamers can be heat eluted with integrated electrodes (yellow) and then electrophoretically transported via wire electrodes across a gel-filled channel (orange) into an isolated chamber (right blue chamber) for further processing Reprinted with permission from Kim et al., Sens Actuators, A 195, 183–190 (2013) Copyright 2013 Elsevier (b) A microfluidic device which utilizes and manipulates target-bound magnetic beads Beads and nucleic acids are added to the central incubation chamber and mixed via integrated microvalves, mixers, and pumps The beads are then held in place using an external magnet and washed of unbound nucleic acids by transporting fluid from the wash chamber to the waste chamber Finally, PCR reagents are pumped from the reagent chamber into the incubation chamber where target-bound aptamers are amplified using micro-heaters and temperature sensors (shown in yellow) for thermocycling Reprinted with permission from Huang et al., Biosens Bioelectron 25(7), 1761–1766 (2010) Copyright 2010 Elsevier (c) A high-throughput sequencing technique which can identify tight binding nucleic acid sequences A microfluidic flow cell and automated fluidic and optical components allow a library of nucleic acids to be sequenced Flowing varying concentrations of fluorescently labeled target molecules through the flow cell and imaging the bound-target intensity allow a binding affinity to be determined and assigned to each nucleic acid sequence The fluorescence images show the locations of individual DNA sequence clusters (top), the location of bound target molecules (middle), and their overlay (bottom) Reprinted with permission from Nutiu et al., Nat Biotechnol 29(7), 659–664 (2011) Copyright 2011 Macmillan Publishers Ltd functionalized beads Bound aptamers are then thermally eluted and transported across the gel into the adjacent chamber via electrophoresis This process can be fluorescently monitored, optimized, and repeated many times if necessary, to further select, store, and concentrate aptamers in the adjacent chamber free from contamination from the other processes taking place in the main chamber The isolation chamber can be used to further integrate and automate amplification steps By capturing transported aptamers onto new beads, integrated heaters and temperature sensors can then be used to thermocycle the chamber in the presence of amplification reagents.128 This device can also be used to perform selections to captured cells A similar device was designed to integrate cell culture and simple valving onto the microfluidic chip to fully integrate the selection process and allow multiple cycles of cell selection to be performed entirely on-chip.129 Interestingly, temperature-dependent selections at 37  C generated aptamers which showed maximum binding at this temperature130 and integrating cell culture into the chip’s selection chamber utilized the integrated heaters and sensors to maintain a temperature of 37  C at all times This demonstrates the ability to generate aptamers which may function optimally at physiological temperatures Another microfluidic device was demonstrated that integrates all of the steps required for SELEX This device utilizes magnetic beads to retain and manipulate target-bound This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-14 K Szeto and H G Craighead aptamers and uses integrated microscale pumps, mixers, and valves to introduce and direct fluids (Figure 7(b)) In addition, integrated microheaters and microtemperature sensors are used to perform on-chip thermocycling for PCR amplifications of the enriched aptamers; and all external controllers for the flow, valves, and other automated protocols are integrated onto a small hand-held system.131,132 Recently, this device was improved to accommodate the transport of enriched and amplified pools with additional reagents allowing multiple cycles of SELEX to be performed continuously and automatically, completely eliminating manual procedures.133 This device has also been similarly adapted to perform selections to viruses134 and cells.135 By integrating separate reagent chambers for target and control cells which are coated with magnetic beads, a two-selection process (positive followed by negative SELEX) utilizing these microfluidic devices allows for almost complete automation of the entire SELEX process for cell specific aptamers on-chip Recently, high-throughput sequencing technologies have been modified to perform affinity measurements with fluorescently labeled targets (Figure 7(c)) Although not demonstrated as a selection technology, second generation sequencing systems already integrate programmable and automated fluidics that can provide various reagents to a microfluidic chip where nucleic acids are assembled on the chip’s surface and their sequences determined fluorescently This is achieved through integrated optics and detectors that can distinguish different bases using unique fluorescent labels After sequencing, fluorescently labeled target molecules are injected at various concentrations and the binding intensity is measured and co-localized with each sequence read.136–138 These methods, called HiTS-FLIP for DNA (HiTS-Fluorescent Ligand Interaction Profiling) and HiTSRAP (-RNA Affinity Profiling) or RNA-MaP (RNA on a Massively Parallel array) for RNA, have been used to perform hundreds of millions of independent affinity assays, measuring the binding affinity for every read and completely eliminating the trial and error identification of high affinity aptamers Although similar to microarrays, these methods can sample orders of magnitude more nucleic acids and not require prior knowledge or design of the library or the enriched pool to be screened In addition, these sequencing platforms contain multiple channels on each chip allowing for the possibility to multiplex with additional targets A possibility for these systems is to use them to identify aptamers to targets without performing affinity selections at all (i.e., cycles of SELEX) as these sequencing methods can already probe similarly sized (or larger) libraries as some of the selection technologies discussed in this review In cases such as these, the elimination of non-binding nucleic acids may be unnecessary, and the starting library may be assayed directly for potential aptamers In this case having not been biased and enriched for any particular target, the sequenced library can be used to measure affinity profiles for multiple targets in serial injections Ultimately, these assays could be used to characterize the SELEX process itself, and the usefulness of other quantitative methods for identifying aptamers, such as multiplicity or enrichment, as well as to model their dynamics between selection cycles Perhaps Appl Phys Rev 1, 031103 (2014) most interestingly, they provide a window into the (effective) affinity distribution of libraries and pools, which is a tremendously useful tool for probing the accuracy of theoretical SELEX models that require this information As sequencing technologies improve and become even more sensitive, the utility of technologies such as HiTS-FLIP, HiTS-RAP, and RNA-MaP will become even more apparent CONCLUSIONS AND FUTURE PROSPECTS A significant amount of progress has been made in the last two decades resulting in more efficient aptamer selections, but the lack of availability of aptamers still limits their large-scale application New technologies are continuing to improve selection efficiencies and reduce the effort needed to obtain new aptamers Future work will no doubt continue to integrate, miniaturize, and automate selection steps, providing a variety of different devices and technologies from which researchers can choose Ultimately, no single technology is superior for all applications and each has limitations that must be considered for a particular application For example, modifications to the nucleic acids and target molecules, such as affinity tags or fluorescent labels, can allow more selection techniques to be used, which result in highly stringent selections, significantly reduced reagent consumption, and the ability to manipulate and monitor binding interactions during the selection process However, modifications can also introduce bias into the binding, and modifications for immobilizing molecules can result in steric hindrance or other unintended binding consequences, such as enhanced non-specific or even specific binding, which are not present in solution-based selections Combining the advantages of several technologies discussed may be an approach for further improving selection efficiencies, while minimizing each technology’s unique sources of background binding, nucleic acid sequence bias, or other undesirable artefacts In addition, other than using stringent or long washing conditions,55 incorporating thermodynamic thresholds for binding and dissociation (although not yet commonplace) may allow for better discrimination between higher and lower affinity aptamers For example, using salt or thermal gradients to elute bound sequences decreases their effective affinity (see Eq (11)) and preferentially releases more weakly bound sequences As the temperature T increases, or the salt concentration increases (causing a decrease in the free energy of binding DG), the effective dissociation constant increases such that only the tightest bound molecules remain Kd ¼ eÀDG=kb T : (11) These methods have been used in simple selection modes to rapidly isolate better binding aptamers in as little as one cycle of SELEX.139–142 Several technologies can perform aptamer selections in a single cycle (or a single “round” of multiple cycles with no amplifications), completely eliminating the majority of the tedious cyclic processes of the traditional SELEX method These are not only fast and efficient but also allow selections to be performed with modified or non-natural nucleic acid This article is copyrighted as indicated in the article Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions Downloaded to IP: 137.222.114.242 On: Mon, 24 Nov 2014 10:59:57 031103-15 K Szeto and H G Craighead libraries that cannot be amplified In addition, minimizing amplifications also minimizes amplification-related bias from pools.143,144 However, unless highly degenerate libraries are used, “enriched” aptamers are likely represented in the final pool with the same or similar frequency (i.e., copy numbers of 1) as lower affinity or even background binding sequences This limitation can be partly alleviated by replacing typical cloning and Sanger sequencing with highthroughput sequencing This allows millions of nucleic acids to be sampled so that bioinformatic techniques can find consensus sequences and conserved secondary structures with great statistical power In contrast, basic cloning/sequencing typically allows only a few dozen nucleic acids to be sampled and is most reliable only when an individual aptamer has copy numbers that dominate the pool However, much more information and confidence can be obtained from sequenced pools that have many different nucleic acids with sufficiently diverse copy numbers that allow population metrics, such as multiplicity or enrichment, to help identify the highest affinity aptamers This places the single-cycle selection methods at odds with newly developed bioinformatic tools, and future methods (where possible) may need to consider maximizing the efficiency of selections with the utility of high-throughput sequencing Additional process characterization has the possibility for improving the selection results for a range of selection techniques Recent studies have shown that in certain technologies, many of the core and intuitive assumptions of the SELEX model and basic binding kinetics are not well supported by the binding results.36,37,60–62,66,67,75 This is likely due to the added complexity of the more sophisticated technologies and immobilization schemes used Selection techniques are currently evaluated primarily on the number of cycles needed and/or on the binding affinity of the resulting aptamer However, the majority of parameters such as times, flow rates, volumes, and concentrations are not typically discussed or understood in many technologies, and there is currently no system by which each can be calibrated to assess its true effectiveness in finding high affinity aptamers Fair comparisons among all the available technologies are hindered due to differences in the selected targets; the library type, size, and origin; as well as the sequencing and analysis methods used These variations obscure the true advantages and disadvantages of each selection technology A standardized target-library system would allow a more direct and fair comparison between methods, and high-throughput sequencing and analysis would allow for more statistically sound metrics to be generated compared to the successful selection or reselection of a single high affinity aptamer, which is susceptible to the stochastics of binding These important issues are valuable opportunities for engineers and physicist to continue to contribute toward our understanding of in vitro aptamer selections in order to improve their design and execution for specific applications Already, significant advancements have been made through clever engineering and the application of non-traditional instrumentation, providing researchers with access to a wider range of physical principles by which to affect the binding and enrichment of high affinity aptamers New sophisticated Appl Phys Rev 1, 031103 (2014) technologies and improved analysis methods have helped to reduce aptamer selections to only a few cycles or less However, new efforts are needed to not only develop new and advanced selection technologies but also optimize 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PHYSICS REVIEWS—FOCUSED REVIEW Devices and approaches for generating specific high- affinity nucleic acid aptamers Kylan Szeto and Harold G Craighead School of Applied and Engineering Physics, Cornell... target-bound nucleic acids and allows unbound nucleic acids to pass through This process is fast and straight forward, and it is one of the most common partitioning methods used for selecting aptamers. .. molecules based on nucleic acids can address the limitations of antibodies and provide new affinity reagents Polymers of nucleic acid molecules have the ability to base-pair and form complex three