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Direct Selection of Fluorescence-Enhancing RNA Aptamers Michael Gotrik,†,‡,∥ Gurpreet Sekhon,‡,∥ Saumya Saurabh,§ Margaret Nakamoto,‡ Michael Eisenstein,‡ and H. Tom Soh*,‡ †

Materials Department, University of California − Santa Barbara, Santa Barbara, California 93108, United States Department of Electrical Engineering and Department of Radiology, Stanford University, Stanford, California 94305, United States § Department of Developmental Biology, Stanford University, Stanford, California 94305, United States ‡

S Supporting Information *

ABSTRACT: RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinitya characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date. In this work, we describe a method for converting DNA libraries into “gene-linked RNA aptamer particles” (GRAPs) that each display ∼105 copies of a single RNA sequence alongside the DNA that encodes it. We then screen large libraries of GRAPs in a high-throughput manner using the FACS instrument based directly on their fluorescenceenhancing properties. Using this strategy, we demonstrate the capability to generate fluorescence-enhancing aptamers that produce a variety of different emission wavelengths upon binding the dye of interest.

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molecule.14−18 As one example, Filonov et al. discovered the Broccoli aptamer through expression of an RNA library in E. coli, followed by incubation with DFHBI and fluorescence partitioning of the brightest cells via fluorescence-activated cell sorting (FACS).14 This powerful approach offers the advantage of directly selecting for aptamers that are stable in vivo and fluoresce brightly in the cellular environment upon binding to the dye. In this work, we describe a complementary, FACS-based screening strategy that uses synthetic particles to rapidly screen individual RNA aptamers based on the fluorescence emission profile generated upon excitation of an aptamer-dye complex. Because the screen is performed entirely in vitro, our method enables screening of fluorescent RNA over wider experimental conditions (e.g., pH, temperature, ionic concentration), as well as for cytotoxic or nonmembrane-permeable dyes for applications beyond cellular imaging. This method builds on the particle display method described previously by our group for screening DNA libraries19−21 and, for the first time, extends it to RNA. We achieve this by converting a randomized DNA library into monoclonal gene-linked RNA aptamer particles (GRAPs), which couple RNA aptamers with their encoding DNA on the surface of monodisperse polymer beads. These GRAPs are then incubated with the target dye and screened via

INTRODUCTION Genetically encoded fluorescent proteins such as green fluorescent protein (GFP) have become an indispensable tool in molecular biology, and researchers today have access to a diverse array of protein-labeling strategies for studying numerous in vivo processes.1 However, there are relatively few methods for directly observing an equally important biomolecule: RNA. While RNA sequences can be selectively labeled through hybridization to a fluorescent probe,2 a more versatile method entails fusing the cellular RNA of interest to an RNA aptamer that generates a strong fluorescent readout upon binding a nonfluorescent small-molecule dye. For example, the Spinach aptamer3 enhances the fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and has enabled direct observation of small-molecule cellular metabolites,4−9 intracellular RNA localization,3,10,11 and the dynamics of cellular processes.3,8,9,12 However, the discovery of novel fluorogenic RNA sequences is difficult using conventional SELEX-style methods. This is because SELEX relies on the enrichment of RNA aptamers based on their affinity for the dye, rather than their actual fluorogenic properties. Previous selection efforts have shown that the binding affinity of an aptamer correlates poorly with fluorescence enhancement.3,13,14 To address this limitation, several groups have recently devised alternative methods for selectively identifying RNAs that can produce a fluorescent signal upon binding to an otherwise nonfluorescent dye © XXXX American Chemical Society

Received: October 8, 2017

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DOI: 10.1021/jacs.7b10724 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society FACS based on their fluorescence emission profile. GRAP display offers several advantages. First, it enables screening at a throughput of ∼5000 particles per second, which is much faster than previously described in vitro screening methods.14,17,18 In addition, our approach allows us to isolate aptamers that exhibit an optimal combination of target affinity and fluorescenceenhancing properties, rather than selecting solely on the basis of either alone. Finally, GRAP display allows us to profile the fluorescence emission of individual RNA aptamers so that we can selectively isolate dye-binding aptamers on the basis of the wavelength at which they fluoresce. As a proof of concept demonstration, we performed two screens against malachite green. We chose malachite green because it naturally exhibits low fluorescence in solution22 (quantum yield of 7.9 × 10−5), and because Wilson and Grate have previously isolated a fluorescence-enhancing aptamer for this dye (MGA) via a conventional SELEX strategy. MGA produces a 2360-fold fluorescence enhancement relative to unbound malachite green22,23 (quantum yield of 0.187), which is among the highest fluorescence enhancement reported to date for an aptamer. Within three rounds of GRAP display, we were able to isolate RNA aptamers that produce considerably brighter fluorescence than MGA, and also to select for functionally distinct aptamer subsets that emit at relatively blue- or red-shifted wavelengths upon binding malachite green. These aptamer should prove immediately useful for in vitro sensing applications, and with further testing, could prove useful for in vivo labeling as well. More generally, this work also demonstrates that the improved speed and efficiency delivered by particle display relative to standard SELEX19−21 are now accessible for RNA aptamers as well as DNA aptamers.

Figure 1. Gene-linked RNA aptamer particle (GRAP) display. A random DNA library is subjected to emulsion PCR under conditions where each emulsion contains no more than a single DNA template (step 1), yielding monoclonal aptamer particles that each display thousands of copies of a single DNA sequence. These are washed and encapsulated in a second emulsion to undergo a transcription reaction (step 2). Newly transcribed RNAs directly hybridize with DNA “capture strands” displayed on the bead surface. The resulting GRAPs are incubated with a nonfluorescent dye (step 3), and GRAPs that produce a fluorescent signal upon binding this dye are separated from those that do not using FACS (step 4). The selected particles are then directly PCR amplified (step 5) for use in further rounds of GRAP display, or subjected to sequencing and further characterization (step 6).

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RESULTS Overview of GRAP Display. GRAP display (Figure 1) enables us to interrogate large numbers of individual RNA aptamer candidates based directly on their ability to enhance the fluorescence of a nonfluorescent dye. GRAP display is based on the particle display method previously described by our group,19−21 in which we use emulsion PCR to convert libraries of aptamers into monoclonal “aptamer particles”. We begin the GRAP display process by covalently modifying the beads with equivalent amounts of a DNA forward primer and a poly-T capture oligo, which is terminated with a 3′ inverted deoxythymidine to inhibit extension during PCR (see Table S1 for sequences of all oligos used and Figure S1 for a more detailed illustration of this process). These beads undergo two consecutive emulsion reactions under stoichiometric conditions such that only 15−30% of beads are coated with RNA, thereby ensuring that each GRAP is monoclonal.19 First, we use emulsion PCR to transform the solution-phase aptamer library into gene particles that display thousands of copies of a single DNA sequence on their surface (step 1). These particles are washed to remove excess reagents, reaction byproducts, and free DNA before being reintroduced into emulsions containing in vitro transcription reagents. In the second emulsion reaction (step 2), each gene sequence undergoes transcription to produce an RNA that includes a 3′ 25-base poly-A tail. This tail allows the transcribed RNA to directly hybridize to the terminated poly-T capture oligonucleotides displayed on the same particle, and ensures that all RNA remains physically associated with the same particle as its encoding gene. This produces GRAPs that display approximately 105 copies of a single RNA sequence and its parent DNA on their surface, and

ensures that the genes encoding RNA sequences of interest can be readily recovered for further screening or analysis. Next, we incubate these monoclonal GRAPs with the target dye of interest (step 3) and measure each particle’s fluorescence signal (Fλ) using FACS (step 4). If the Fλ of a measured GRAP exceeds a signal threshold defining the “sort gate”, it is collected and directly subjected to PCR amplification in preparation for further screening (step 5). Typically, the target concentration is adjusted prior to FACS screening such that only a small fraction of the particles (e.g., 0.05−0.20% of the total population) resides above the sort gate threshold, and the brightest ∼0.01% of events are collected for further screening. Pool enrichment is monitored by comparing the fraction of beads that exceeds the sort gate threshold at a given dye concentration across subsequent rounds of selection. If a higher fraction of beads produces a fluorescence signal exceeding this threshold relative to the previous round, they are subjected to an additional round of screening at a reduced dye concentration in order to increase selection stringency. This process is repeated until no further enrichment is observed, at which point the pools are subjected to high-throughput sequencing (HTS) and candidate aptamers are identified for further characterization (step 6). Our technique differs from conventional SELEX-style experiments that preferentially enrich aptamers based on their affinity to the target molecule without offering any insight into their fluorescence-enhancing capabilities. Since aptamer affinity correlates poorly with fluorescence enhancement in this context,3,13,14 conventional SELEX can lead to undesired enrichment of aptamers with poor fluorescent properties. In contrast, GRAP display allows us to directly profile and isolate B

DOI: 10.1021/jacs.7b10724 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society aptamers based on their fluorescence-enhancing properties as well as their ability to bind the target dye. The Fλ measured via FACS from a given GRAP upon binding malachite green is a product of a number of factors, including aptamer affinity (Kd), target dye concentration ([T]), and the complex’s quantum yield (ϕf) as described by the following equation: Fλ ≈ ϕf

[T ] Sλ [T ] + Kd

(1)

where Sλ is the number of photons emitted in the measurement window. Thus, the aptamers isolated from GRAP display possess a combination of high quantum yield, high affinity to the dye (low Kd), and abundant emission at the desired wavelength. Screening of a Structured Fluorescence-Enhancing RNA Aptamer Library. As a preliminary demonstration, we designed an aptamer library that was structured around a known, minimized fluorescence-enhancing malachite greenbinding motif derived from MGA22 (see Table S1 for sequences of all oligos used, Figure S2 for library design rationale). In this design, the destabilized malachite greenbinding motif was inserted between two 15-base random regions, and the resulting library was subjected to screening with GRAP display in order to see whether we could successfully isolate variants with improved fluorescence or binding affinity. The theoretical diversity of this library is much greater than the throughput that can realistically be screened with FACS (typically ∼108 sequences). However, incorporating a large region of the known malachite green-binding motif greatly improves the likelihood that a sequence will exhibit some degree of binding and fluorescence enhancement. Thus, we directly converted the structured library to GRAPs as described above, and performed three consecutive rounds of GRAP display. In order to maintain a high selective pressure, we lowered the concentration of malachite green in each round such that, typically, 0.05−0.20% of GRAPs resided within the sort gate threshold in the 670 ± 30 nm emission channel. We then collected the brightest ∼0.01% of all GRAPs screened in each round. In the first round, we incubated our library with 50 nM malachite green (Figure 2), after which we reduced the dye concentration to 1 nM in Round 2 and 400 pM in Round 3. The mean fluorescence signal of the GRAP pool greatly increased over the course of sorting; for example, 21.8% of GRAPs exhibited above-background fluorescence in the Round 1 pool in the presence of 50 nM malachite green, compared to 0.22% in the starting pool at the same concentration. The level of enrichment decreased in subsequent rounds using lower target concentrations; after Round 3, no further enrichment was observed and all three pools were sequenced. GRAP Display Generates Brighter, Higher-Affinity Aptamers than MGA. We identified 1.5−2.4 × 106 sequences from each pool. To narrow these to a number suitable for further characterization, we first obtained a reads-per-million (RPM) value for each unique sequence and identified the five most abundant sequences in the Round 3 pool. Next, we determined a fitness value (i.e., the ratio of a sequence’s current round RPM over its previous round RPM) for all sequences that were represented by >20 copies in the raw sequencing data. Previous studies have shown that such enrichment metrics are a better predictor of performance than copy number

Figure 2. Summary of GRAP display screening with a structured library. FACS plots show fluorescence signal from each GRAP pool at the designated malachite green concentration before (left) and after (right) screening. We did not see any further increase in the fluorescence-enhancing subpopulation of GRAPs after a third round of screening, and no additional screening was performed.

alone.24−26 We then identified the five highest-fitness sequences in the Round 3 pool. In total, we chose 10 sequences for further characterization (Table S2). We then compared our sequences to the full-length and minimized MGA aptamers.22,23 Briefly, we performed a titration from 10 nM to 10 μM malachite green using 100 nM RNA, and measured the fluorescence response of each sequence using a Tecan M1000 plate reader. Next, we fitted the response profile to an equation for 1:1 complexation to yield dissociation constants (Kd) and fluorescence enhancement values, which were determined relative to the full-length MGA aptamer (see Experimental Section). It should be noted that newly transcribed RNA generated during GRAP display incorporates a 3′ 25-base poly-A tail to allow hybridization to the beads, and it is possible that this tail is partially incorporated into the properly folded aptamer. Here, we characterized all sequences without the poly-A tail, as the minimal MGA-derived active region of these sequences is known and was not predicted by Mfold software to interact with the poly-A tail. By plotting the fluorescence enhancement and Kd of each sequence (Figure 3), we can see that GRAP display allows us to identify sequences that outperform both the minimal MGA domain and full-length MGA (Table S3). All 10 of the aptamers we identified enhanced the fluorescence of malachite green. However, MG-D10 and the minimized MGA motif had an affinity lower than could be determined with our assay, and so neither brightness nor Kd are reported. Aptamers MG-D3 and MG-D7 exhibited stronger fluorescence enhancement than MGA (around 1.6-fold and 1.2-fold, respectively). Indeed, to the best of our knowledge, MG-D3 exhibits the greatest magnitude of fluorescence enhancement reported for any RNA aptamer to date. C

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Figure 3. Characterization of sequences identified from our structured library. A plot of dissociation constant (Kd) and fluorescence enhancement (fold-increase in fluorescence over background) measurements for the aptamer sequences obtained via GRAP display relative to the well-characterized malachite green aptamer MGA. The Kd of the minimal MGA domain exceeded what could be measured in our assay, and therefore is not shown here.

De Novo Discovery of Novel Fluorescence-Enhancing RNA Aptamers for Malachite Green. We subsequently demonstrated that GRAP display can efficiently discover novel malachite green-binding aptamer sequences by using an unstructured, random library (see Experimental Section and Table S1 for details). While traditional SELEX is capable of addressing a library diversity upward of ∼1014 sequences per round, the throughput of GRAP display is limited by that of the FACS instrument. We therefore needed to reduce the diversity ̈ library to a scale that could be effectively analyzed of our naive using FACS in ∼10 h (∼108 sequences). Similar to other FACS-based aptamer selections,14,19 we performed two rounds of affinity-based, pre-enrichment SELEX so as to reduce the library size while still maintaining the presence of functional sequences. This pre-enriched library was then converted into GRAPs and incubated with 10 μM malachite green before undergoing FACS screening (Figure 4A). We isolated the brightest GRAPs that fluoresced at wavelengths greater than malachite green’s primary excitation peak (618 nm). Specifically, we collected the brightest ∼0.01% of beads from three emission channels: 670 ± 30 nm, 730 ± 45 nm, and 780 ± 60 nm. These emission channels were chosen with the intention of identifying aptamers with a variety of distinct fluorescence profiles, as described in greater detail below. The GRAPs collected from all three emission channels were then pooled prior to PCR amplification in order to prepare a single library for the next round of GRAP display. Over the following rounds, we made the screening conditions more stringent by decreasing the concentration of malachite green from 10 μM in Round 1, to 1 μM in Round 2, and to 100 nM in Round 3. After three rounds of screening, the average fluorescence intensity of the displayed aptamers increased dramatically (Figure 4A), with a far larger fraction of GRAPs residing above the sort gate thresholds in the Round 3 pool (8.85−15.2%) compared to the starting pool (0.15−0.19%), even in the presence of a 100-fold reduced concentration of malachite green. Previous work has shown that different aptamers can generate distinct emission profiles upon binding to the same target molecule.3 To explore whether we could differentiate subpopulations of aptamers that emit at different wavelengths, we compared the relative Fλ signals of the Round 3 pool across

Figure 4. GRAP display screening with a randomized library. (A) We performed three rounds of screening, with sort gates designating events exhibiting the brightest fluorescence above background in the 670 ± 30 nm, 730 ± 45 nm, and 780 ± 60 nm gates. Sorted particles (the brightest ∼0.01%) were pooled, amplified, and used to produce GRAPs for the next round of screening. We observed no significant increase in fluorescence from the Round 3 pool after incubation with 10 nM malachite green (data not shown), and performed no additional screening. (B) We separated the Round 3 pool into spectrally shifted subpopulations with relatively higher mean 780 ± 60 nm (Round 3-R, top) or 670 ± 30 nm (Round 3-B, bottom) fluorescence.

the 670 ± 30 nm and 780 ± 60 nm emission channels (Figure 4B). We found that after incubating with 1 μM malachite green, we could readily separate the Round 3 pool into distinct blueshifted F670±30nm > F780±60nm (Round 3-B) and red-shifted F780±60nm > F670±30nm (Round 3-R) pools. We then performed HTS and obtained 2.4−4.2 × 106 sequences from each of the Round 2, Round 3, Round 3-B, and Round 3-R pools. As described above, we determined RPM values for all sequences, selecting the five most highly represented sequences in the Round 3, Round 3-B, and Round 3-R pools. This union comprised five sequences from Round 3 and three sequences from Round 3-R; the five most abundant sequences in Round 3-B were identical to those from Round 3. Next, we determined a fitness value for all sequences D

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Journal of the American Chemical Society as described above. Many high-fitness sequences in each round were similar to one another with the exception of a one- or twobase mismatch, insertion, or deletion. For example, sequence R3-B-3 differs from sequence R3-4 by a single point-mutation, and 95 of the 100 highest-fitness sequences in the Round 3-B pool were highly similar to R3-4 based on this standard. We looked at the 100 most enriched sequences in each pool and chose the five highest-fitness, nonsimilar sequences (where possible) from the Round 3, Round 3-B, and Round 3-R pools. This union comprised five sequences from Round 3, four sequences from Round 3-B, and five sequences from Round 3R. In total, we selected 22 sequences for further functional characterization (Table S4). We then measured the binding affinity and fluorescence enhancement for each of our selected aptamers using MGA as the standard, as described above. Briefly, we titrated the 22 aptamers with various concentrations of malachite green ranging from 10 nM to 10 μM, and measured the fluorescence response at each concentration. Because we were unable to predict the contribution of the poly-A tail region to aptamer function, we determined the Kd and brightness values (relative to MGA) both with and without the tail, and report here the data for only the more functional of the two (Table S4). The sequence and functional characteristics of all 22 aptamers are summarized in Table S5. In total, 21 of the 22 selected sequences generated measurable fluorescence (ranging from 18- to 4273-fold enhancement above background) after incubation with malachite green, and exhibited affinities spanning nearly 2 orders of magnitude (Figure 5A). Only R3-3 exhibited no activity in our assays. This is in striking contrast to a previous SELEX-based effort, in which 2 μM) such that they were outside the quantifiable range of our assay; as a result, we were unable to calculate accurate Kd and fluorescence enhancement values. Three aptamers exhibited higher binding affinity than MGA, but were dimmer than this previously described aptamer. On the other hand, R3-B-4 was ∼80% brighter than MGA at saturation despite having a ∼6-fold reduced affinity for malachite green (Figure 5C). The wide diversity of aptamer properties we

Figure 5. Characterization of sequences isolated from de novo GRAP display screening. (A) Sequences identified via GRAP display exhibit a range of affinities for malachite green. R3-8 had the highest affinity, R3-1 was the most common sequence in the Round 3 and Round 3-R pools, and R3-B-4 exhibited the brightest fluorescence enhancement, but with lower affinity relative to MGA. (B) Affinity is poorly correlated with fluorescence enhancement, and GRAP display can enrich sequences that would be lost with purely affinity-based selection. Sequences that were negatively enriched (unfilled shapes) in Round 3-R (red) and Round 3-B (blue) relative to the Round 3 pool exhibit higher affinity, but poorer fluorescence-enhancing capabilities. In contrast, those that were positively enriched in the Round 3-R and Round 3-B pools (filled shapes) generally exhibit much brighter fluorescence. (C) Sequence R3-B-4 exhibits higher brightness at saturation (quantum yield) than MGA. RNA concentration is 100 nM.

obtained, coupled with the poor correlation between aptamer affinity and brightness highlights the advantages of using GRAP display to simultaneously screen for both target affinity and E

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Journal of the American Chemical Society fluorogenic properties, rather than merely affinity as is done with traditional SELEX. GRAP Display Enables Selective Enrichment of Aptamers Based on Emission Spectra. By selectively gating in different emission channels, we were able to screen for aptamers that exhibit distinct fluorescence emission profiles upon binding the same dye. As described above, we sorted the Round 3 GRAPs based on their relative fluorescence in the 670 ± 30 nm and 780 ± 60 nm emission channels, and separated this pool into blue-shifted (Round 3-B) and red-shifted (Round 3-R) subpopulations (Figure 4B). We initially expected to identify only two emission profiles within the Round 3-B and Round 3-R pools, but instead identified subsets of aptamers with a variety of distinct excitation and emission maxima (Table S5). The emission maxima for the characterized Round 3 sequences ranged from 649−675 nm, consistent with the collection of all aptamer particles that fluoresce at wavelengths above the primary excitation peak of malachite green (618 nm). In contrast, the sequences enriched in Round 3-B exhibited a much narrower range of emission maxima ranging from 649− 654 nm, while all characterized sequences enriched in Round 3R shared an emission maximum of 675 nm. Notably, this unique emission profile appears to result from a short, 15-bp motif conserved among these sequences (Table S6). The excitation and emission spectra for the most abundant sequences in the Round 3-B and Round 3-R pools (R3-4 and R3-1, respectively) are shown in Figure 6 alongside the MGA aptamer. These results show that GRAP display can effectively isolate aptamers that produce distinct emissions profiles upon binding the same dye.

and emission wavelength properties. The key to our approach is that it enables us to directly screen large numbers of individual aptamers on the basis of both dye-binding affinity and fluorescence emission, as opposed to selecting solely on the basis of affinitya property that is known to correlate poorly with an aptamer’s fluorescence-enhancing capabilities.3,13,14 This is consistent with previous findings, which have shown that fluorescence-based selection yields superior fluorogenic aptamers.14,15,17 Using our method with a library structured around the known MGA sequence, we identified numerous aptamers that greatly outperformed their parent sequence in terms of both affinity and brightness. This enhanced performance relative to MGA could be a product of superior folding, thermodynamic properties or other biochemical characteristics, and it will be informative to explore this structure−function relationship in future work. Additionally, after just two rounds of preenrichment followed by three rounds of GRAP display, we obtained a novel aptamer that generated 4273-fold fluorescence enhancement upon binding to malachite greena considerable improvement over MGA, which previously exhibited the highest reported fluorescence enhancement for an RNA-dye complex. Although none of the de novo-selected aptamers examined here outperformed MGA in terms of both affinity and brightness simultaneously, the success of our experiments with the structured libraries suggests that it should be feasible to achieve considerable further optimization of these aptamer sequences via directed evolution using GRAP display. Lastly, we demonstrate the capability to select subpopulations of aptamers that emit at different wavelengths upon binding to the same dye, with emission maxima ranging from 649−675 nm. Previously, sequence-specific fluorescence profiles have been observed for fluorogenic aptamers that recognize a common dye target,3 and Song et al. have recently shown that directed evolution of the Broccoli precursor aptamer using FACS-based screening can yield red-shifted aptamer variants.16 In future selections, we believe the capability to generate aptamers that emit at specific wavelengths for a wide range of fluorogenic substrates will be valuable for many imaging applications. Given that FACS instruments have become available in many research settings and that GRAP display relies only on widely available reagents and equipment, we believe that our method could be readily utilized by the broader research community to build an extensive toolbox of fluorescent RNA sensors. Our previous work has shown that particle display-based methods dramatically reduce the number of rounds required to isolate high quality DNA aptamers relative to conventional SELEX, while also increasing the likelihood of successful screening against challenging targets.19−21 On the basis of the RNA molecules obtained after three rounds in this study, we anticipate that the same should hold true for particle displaybased RNA aptamer selection. As such, GRAP display should offer a powerful general tool for the discovery of conventional RNA aptamers as well as other classes of functional RNA molecules.

Figure 6. Excitation and emission profiles of sequences with distinct fluorescence spectra. We isolated multiple fluorescence-enhancing aptamers with excitation (dashed) and emission (solid) profiles that are markedly distinct from that of MGA (black). R3-4 (blue) and R31 (red) respectively represent the furthest blue-shifted and red-shifted spectra identified in our screen.

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CONCLUSION Fluorescence-enhancing aptamers have the potential to accelerate the study of RNA biochemistry, localization and processing, and to greatly expand the utility of RNA-based materials in fields such as bioimaging and biosensing. To this end, we describe GRAP display, a high-throughput, FACSbased in vitro RNA aptamer screening strategy that we have used to isolate multiple fluorogenic aptamers within just a few rounds of screening. Using this process, we can obtain a diverse collection of aptamers exhibiting distinct affinity, brightness,

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EXPERIMENTAL SECTION

Immobilization of Malachite Green for SELEX. We immobilized malachite green (MG) isothiocyanate (Molecular Probes) on the surface of 2.7-μm Dynabeads M-270 amine beads (Invitrogen). The beads were activated in 100 mM NaHCO3 buffer, pH 8.5, and suspended in 100 mM NaHCO3 buffer, pH 9. MG isothiocyanate was added to the beads at a final concentration of 500 μg/mL and F

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Journal of the American Chemical Society incubated with rotation overnight. The supernatant was removed and the beads were washed by resuspending them in pure dimethyl sulfoxide (DMSO), heating to 70 °C, and removing the darkened supernatant. The washing process was repeated until the DMSO remained clear after heating. After washing, the beads were visibly darkened, indicating successful functionalization. Washed beads were suspended in TNaTE buffer (10 mM Tris, 200 mM NaCl, 0.01% Tween-20, 0.1 mM EDTA) and stored at 4 °C. DNA Preparation. The single-stranded DNA libraries and all primers used were synthesized by Integrated DNA Technologies (see Table S1 for all oligonucleotides used). Modified oligos were purified using HPLC, while all others underwent standard desalting. The libraries used for this selection were adapted from a previously reported RNA aptamer library.27 The structured DNA library contained a destabilized, minimized MG-binding motif surrounded by two randomized 15-nucleotide (nt) regions that were themselves flanked by conserved regions for transcription and PCR: (5′TAATACGACTCACTATAGGGACACAATGGACG-[N15]CCGACTGGCGAGAGCCAGGTAACGAATG-[N15]-TAACGGCCGACATGAGAG-3′). The naiv̈ e DNA library consisted of molecules containing a randomized 40-nucleotide (nt) region flanked by conserved regions used for transcription and PCR: (5′-TAATACGACTCACTATAGGGACACAATGGACG-[N40]-TAACGGCCGACATGAGAG-3′). The reverse primer (5′-CTCTCATGTCGGCCGTTA-3′) (RP) was synthesized both with and without a 25-nt poly-T tail at the 5′ end for use as a capture strand upon transcription. The forward primer (5′-TAATACGACTCACTATAGGGACACAATGGACG-3′) (FP) contained a T7 promoter region to allow transcription of the expressed aptamers. Additionally, a FP and a 25-nt poly-T capture strand were ordered, featuring a 5′-amino modification separated from the first nucleotide by a 72-atom internal spacer. The amino-modified poly-T was terminated with a 3′ inverted deoxythymidine to inhibit extension by DNA polymerases. Coupling Forward Primer to Particles. 500 μL of 1-μm MyOne carboxylic acid beads (Life Technologies, 1010/mL) were activated by washing once with 500 μL of 0.01 M NaOH and five times with 500 μL ddH2O. The beads were incubated with 1× conjugation buffer (200 mM NaCl, 100 nM imidazole), 20 μM amino-modified FP, 180 μM amino-modified poly-T, and 250 mM 1-ethyl-3-(3diemthylaminopropyl)carbodiimide (EDC) in 50% v/v DMSO overnight at RT with rotation. The beads were then washed twice with 500 μL 0.1 M 2-(n-morpholino)ethanesulfonic acid (MES) buffer (100 mM, pH 4.7) (Pierce Biotechnology) for 10 min with rotation and resuspended in MES buffer. To passivate any unreacted functional sites, we converted unreacted carboxyls into amino-reactive NHS-ester groups in the presence of 250 mM EDC and 100 mM Nhydroxysuccinimide (NHS) in MES buffer for 30 min at RT. The beads were conjugated with 20 mM amino-PEG12 in MES buffer for 1 h at RT. The FP-modified beads were then washed two times with TT buffer (0.05% Tween-20, 50 mM Tris, pH 7.5) for 5 min to quench the reaction, resuspended in 500 μL TNaTE buffer, and stored at 4 °C. To test conjugation efficiency, we incubated two separate batches of 0.2 μL of FP beads with 1 μM FAM-RP-complement and 1 μM FAMpoly-A, respectively, in 100 μL TNaTE buffer for 5 min at room temperature. Each batch of beads was washed once with 100 μL TNaTE buffer, and analyzed using a BD FACSVerse cytometer (BD Biosciences). To confirm that the FP beads were compatible with PCR, we performed PCR with 1 μM FP, 1 μM RP-polyT, 1 nM template DNA, 1× GoTaq PCR Master Mix (Promega), and 1 μL FP beads in 20 μL total volume. The PCR program used was 98 °C for 2 min, followed by 20 cycles of 98 °C for 15 s, 57 °C for 30 s, and 72 °C for 30 s. These beads were then washed three times using 100 μL 100 mM NaOH, washed once with 100 μL TNaTE buffer, incubated with 1 μM FAM-RP-polyT for 5 min at RT, washed once with 100 μL TNaTE buffer, and analyzed on the FACSVerse. Generally, the fluorescence signal after PCR was ∼10-fold lower than for the primer conjugation efficiency test described above. We found that a 72-atom spacer is necessary to separate the amino coupling from the first nucleotide in both the FP and poly-T primer sequences that are displayed on the FP beads. When these

oligonucleotides were conjugated to the beads without a spacer, PCR efficiency was dramatically reduced; we believe this to be a result of steric effects. This was confirmed by conjugating an amino-dPEG24t-butyl ester to the surface of the carboxylic acid beads using EDC in the manner described above, deprotecting the spacer by washing three times with trifluoroacetic acid (TFA), and then using EDC to conjugate amino-modified primers containing no internal 72-atom spacer, and passivating any unreacted carboxyl groups. After comparing the PCR performance of FP beads containing no internal spacer to FP beads containing either the PEG24 spacer or the 72-atom internal spacer used in these experiments. We found that efficient PCR amplification on the surface of the beads was only possible in the presence of a spacer. RNA Library Pre-enrichment. We PCR amplified 1 × 1014 sequences of the starting library with 1 μM FP, 1 μM RP, and 1× Master Mix to prepare a double-stranded library for use in transcription. To minimize PCR bias, we performed only 6 cycles of PCR on a total volume of 8 mL using the following PCR protocol: 98 °C for 2 min, then 6 cycles of 98 °C for 15 s, 57 °C for 30 s, and 72 °C for 30 s. The DNA was separated from the reaction mixture using phenol:chloroform extraction and the aqueous phase was concentrated via n-butanol extraction. The recovered DNA was ethanol precipitated using 2.75 volumes of ethanol and 0.1 volumes 3 M sodium acetate (pH 5.5), and the recovered pellet was suspended in TE (10 mM Tris, 0.1 mM EDTA, pH 7.5) buffer. Transcription of 8 × 1014 DNA sequences was performed in five reaction volumes (100 μL total) with the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific), following the manufacturer’s protocol. After DNase treatment for 1 h, the recovered RNA was purified using a 6% urea-TBE acrylamide gel (National Diagnostics), cut from the gel after UV shadowing, and recovered through the crushand-soak method in DEPC-treated water. The eluent was ethanol precipitated as described above, and the recovered pellet was suspended in TE buffer. In the first round of pre-enrichment SELEX, we heat denatured ∼8 × 1015 RNA molecules for 5 min at 70 °C and quickly placed on ice to cool. The RNA was incubated with ∼1 × 109 MG-coated beads in PBSMT buffer (1× PBS pH 7.4, 5 mM MgCl2, 0.01% Tween-20), washed two times with PBSMT, eluted using PBSMT buffer at 75 °C for 1 min, and reverse-transcribed using cloned AMV reverse transcriptase (Invitrogen) following the manufacturer’s instructions. We amplified the recovered DNA using PCR and transcribed into RNA as described above for use in a second round of pre-enrichment. In the second round of SELEX, we reduced the amount of RNA and target, using 4.5 × 1014 RNA sequences and 6.5 × 107 MG-coated beads. GRAP Synthesis. We generated our GRAPs using emulsion PCR followed by emulsion transcription. The oil phase in both reactions consisted of 95% mineral oil, 4.5% Span 80, 0.45% Tween 80, and 0.05% Triton X-100. For emulsion PCR, the aqueous phase consisted of 1× PCR Master Mix (Promega), 10 nM FP, 1 μM RP-polyT, ∼108 beads, and 1.5 pM template DNA. Water-in-oil emulsions were generated for emulsion PCR by adding 1 mL of the aqueous phase to 7 mL of oil phase in a DT-20 tube (IKA), which was locked into an Ultra-Turrax Device (IKA). The addition was performed dropwise over 30 s while being stirred at 620 rpm for 5 min. We performed PCR under the following cycling conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 57 °C for 60 s, and 72 °C for 60 s. After PCR, the emulsions were collected into an emulsion collection tray (Life Technologies) through brief centrifugation. The emulsions were broken using 3 mL of 2-butanol and transferred to a 50 mL tube. The particles were pelleted by centrifugation at 4000g for 5 min and the supernatant was decanted. The particles were then resuspended in emulsion breaking buffer (BB) (100 mM NaCl, 1% Triton X-100, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) and transferred to a 1.5 mL tube. The beads were sonicated, vortexed, centrifuged for 15 s at 13 000g and placed in a magnetic separator (MPC-S, Life Technologies). The supernatant was removed via pipet and the beads were washed once with 100 μL PBS, once with 100 μL BB, and three times with 100 μL 100 mM NaOH to generate ssDNA and G

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Journal of the American Chemical Society remove noncovalently bound DNA. We confirmed that 20 ± 10% of the particles contained DNA by annealing ∼50 000 beads with 1 μM FAM-RP-poly-T at RT for 5 min. The particles were washed once with 200 μL PBS-T and analyzed using the FACSVerse. To generate double-stranded DNA on the particles, we subjected the particles to one cycle of PCR (95 °C for 2 min, 57 °C for 30 s, and 72 °C for 60 s) using 1× GoTaq PCR Master Mix and 1 μM FAM-labeled RP. The beads were washed twice with 100 μL BB and resuspended in 500 μL TNaTE. Emulsion transcription was performed using the T7 High Yield Transcription Kit. The aqueous phase consisted of 0.4× TranscriptAid Reaction Buffer, 4 mM of each rNTP, 5 mM MgCl2, ∼5 × 107 DNAcoated beads/mL, and 0.4× TranscriptAid Enzyme mix. For the starting pool in both selections, we used 1 mL of aqueous phase and 7 mL of oil phase, and the emulsions were prepared in the UltraTurrax device as described for ePCR. In every subsequent round of emulsion transcription, the emulsions were made in quadruplicate through dropwise addition of 50 μL aqueous phase over 15 s to 450 μL oil phase in 2 mL cryogenic vials (Corning), which were being mixed at 1000 rpm by Teflon flea micro stir bars (Fisher). The reaction vials were stirred for 3 min, then incubated at 37 °C for 6−10 h. The emulsions were kept on ice while pooled, broken using 3 mL of cold 2butanol, and transferred to a centrifuge tube. The particles were pelleted by centrifugation at 4 °C and the supernatant was decanted. The particles were then resuspended in cold BB and transferred to a 1.5 mL tube. The particles were sonicated, vortexed, centrifuged for 15 s at 4 °C and 13 000g, and then placed on an MPC-S. The supernatant was removed by pipet. Further wash steps were performed without centrifugation using cold BB, until all oil in the supernatant was removed and the beads no longer visibly aggregated. To confirm that the particles contained both DNA and RNA and were sufficiently monodisperse, we incubated ∼50 000 aptamer particles in 100 μL TNaTE containing 1 μM Alexa Fluor 647-labeled RP at RT for 5 min, washed once using 100 μL TNaTE, and analyzed using the FACSVerse (see Figure S3 for representative FACS plots of this process). FACS Screening. Prior to screening, we incubated FP beads with MG (Spectrum Chemical) at concentrations ranging from 10 nM to 10 μM in PBSMT to determine levels of background fluorescence and nonspecific binding. All incubations were performed at RT for 30 min. FP beads give an appropriate baseline, because ∼80% of particles after ePCR contain no PCR product and little or no RNA, making them indistinguishable from FP beads. We performed FACS on the Aria II instrument (BD Biosystems), isolating particles that exhibited a fluorescence signal above background (defined as the reference gate) due to MG activation and which also exhibited a strong fluorescence signal from the FAM-labeled double-stranded DNA. This ensured that only particles conjugated to both RNA and its parent DNA were collected and enriched. We incubated and sorted ∼108 particles for the starting GRAP library of both selections. In all later rounds, only ∼107 particles were incubated and sorted at a MG concentration such that 0.05−0.2% of the total population fluoresced above the sort gate threshold, and the brightest ∼0.01% of events were collected. The FACS instrument’s flow rate was adjusted such that ∼5000 events/ second were registered. The collected sample containing GRAPs was concentrated on an MPC-S, excess sheath supernatant was removed, and particles were resuspended in 100 μL PCR mix containing 1 μM FP, 1 μM RP, and 1× Master Mix. PCR was performed on the collected particles using the following protocol: 98 °C for 2 min, then 14−20 cycles of 98 °C for 15 s, 57 °C for 30 s, and 72 °C for 30 s. The number of particles collected for all FACS sorting procedures as well as the sorting conditions and emission channels used for each round are summarized in Table S7. Sequencing Analysis. High-throughput sequencing was performed at the Stanford Functional Genomics Facility (SFGF) using the MiSeq System (Illumina). Using a custom MATLAB script implementing the Bioinformatics Toolbox, the sequence data was analyzed to obtain copy numbers of all sequences. The copy numbers were then normalized to the total number of reads from each pool to obtain a reads-per-million (RPM) value for all sequences in every pool.

We then calculated a fitness value for all sequences that were present in at least 20 copies in the raw data. This value was defined as the ratio of a sequence’s current-round RPM value over the previous-round RPM. Sequences identified from both selection procedures (copy number and fitness value) were chosen as described in the Results section above, and ordered from the Protein and Nucleic Acid (PAN) Facility at Stanford University. RNA Aptamer Synthesis. The identified candidates were ordered as single-stranded DNA oligos and PCR amplified using 1 μM FP, 1 μM RP, 1 nM template DNA, 1× GoTaq PCR Master Mix at a total volume of 100 μL and with the following protocol: 98 °C for 2 min, then 6−10 cycles of 98 °C for 15 s, 57 °C for 30 s, and 72 °C for 30 s. RNA was transcribed using a TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) and following the manufacturer’s protocol. After DNase treatment for 30 min, the recovered RNA was purified using a 6% urea-TBE acrylamide gel (National Diagnostics), cut from the gel after UV shadowing, and recovered through the crush-and-soak method in DEPC-treated water. The eluent was ethanol precipitated as described above, and the recovered pellet was suspended in TE buffer. Fluorescence Characterization. A stock MG concentration was prepared in PBS-T buffer (1× PBS pH 6.1, 0.01% Tween-20) and subjected to serial dilutions from 20 μM to 20 nM in the same buffer. The concentration of MG was measured daily using a UVspectrophotometer (NanoDrop, Thermo Scientific) and calculated with an extinction coefficient of 148 900 cm−1/M at 616.5 nm28 after allowing MG to equilibrate overnight in PBS-T. Assays were prepared in flat-bottomed 96-well MicroFluor 2 plates (Thermo Fisher Scientific) at a final concentration of 100 nM RNA, 5 mM MgCl2, and 0.01% Tween-20, with MG concentrations ranging from 10 nM to 10 μM. Titrations were prepared and measured in triplicate. Wells were excited at 630 ± 20 nm, and emission was measured at 675 ± 20 nm. For each MG concentration measured, an RNA-free standard was measured in triplicate and subtracted from the signal as a baseline. The signals were plotted at each MG concentration and least-squares fit to a 1:1 complexation model using a custom MATLAB script according to the following equation: F = Bmax ×

[T ] [T ] + Kd

where F is the measured fluorescence signal, and Bmax is the fluorescence signal at binding saturation. Quantum yields were obtained by first comparing the integral of the corrected excitation and emission spectra for each aptamer to that of MGA across the regions used for excitation (610−650 nm) and emission (655−695 nm) measurements. These integrals were then used to calculate an adjusted Bmax value for each aptamer, which was then compared to the Bmax measured for MGA (quantum yield of 0.187, or a 2360-fold fluorescence enhancement27) and used to calculate quantum yields. Measurements of Excitation and Emission Profiles. Solutions containing 1× PBS, 5 mM MgCl2, 3 μM MG, and 1 μM RNA at pH 6.1 were prepared for each aptamer, and excitation and emission spectra were measured on a UV fluorometer in a quartz cuvette (Hellma Analytics). The measurements were performed in triplicate, and a baseline corrected average was obtained after subtracting the measurement from an RNA-free standard.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b10724. Library design, SELEX procedure, GRAP synthesis, FACS procedure and sorting results, sequencing results, fluorescence and binding characterization, and information on the minimum red-shifted motif (PDF) H

DOI: 10.1021/jacs.7b10724 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society

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(25) Schütze, T.; Wilhelm, B.; Greiner, N.; Braun, H.; Peter, F.; Mörl, M.; Erdmann, V. a.; Lehrach, H.; Konthur, Z.; Menger, M.; Arndt, P. F.; Glökler, J. PLoS One 2011, 6, 1−10. (26) Hoinka, J.; Berezhnoy, A.; Sauna, Z. E.; Gilboa, E.; Przytycka, T. M. Lect. Notes Comput. Sci. 2014, 8394, 115−128. (27) Miyakawa, S.; Nomura, Y.; Sakamoto, T. RNA 2008, 14, 1154− 1163. (28) Green, F. J. The Sigma-Aldrich Handbook of Stains, Dyes and Indicators; Aldrich Chemical Company, Inc.: Milwaukee, WI, 1990.

AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

H. Tom Soh: 0000-0001-9443-857X Author Contributions ∥

MG and GS contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors appreciate the support from DARPA (N66001-14-24055) and the Garland Initiative. HTS is a Chan-Zuckerberg Biohub investigator.

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