Direct Screening for Cytometric Bead Assays for Adenosine

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Direct Screening for Cytometric Bead Assays for Adenosine Triphosphate Hao Qu, Lu Wang, Jian Liu, and Lei Zheng ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00224 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 12, 2018

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Direct Screening for Cytometric Bead Assays for Adenosine Triphosphate Hao Qu,†,‡ Lu Wang,∗,† Jian Liu,∗,† and Lei Zheng∗,¶ †School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China ‡CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China ¶School of Food Science and Engineering, Hefei University of Technology, Hefei, Anhui 230009, China E-mail: [email protected]; [email protected]; [email protected]

Abstract Cytometric bead assays have caught much attention because of their many exceptional advantages. Unfortunately, the immobilization of existing molecular recognition elements including monoclonal antibodies and aptamers onto solid particles may lead to the function failure of the molecular recognition elements since they are generally obtained in free state. Herein we develop a powerful screening approach for direct and rapid discovery of aptamer based cytometric bead assays (AB-CBAs) by individually measuring the functional activity of every aptamer particles in a library and sorting them at rates of up to 108 particles per hour. The strategy is based on the transformation of molecular libraries into pools of monoclonal aptamer particles so that one individual particle displays ∼ 105 copies of an identical aptamer sequence. Our library design incorporates a two-color fluorescent reporter system in which changes in aptamer

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structure generate an optical readout, such that we can use fluorescence-activated cell sorting to rapidly and selectively separate the individual aptamer particles that exhibit large fluorescent signal change upon target binding. For demonstration, we isolated AB-CBA aptamer particles with high signaling performance for ATP after just 3 rounds of screening. We believe that the rapid and direct screening features of this strategy make it an excellent platform for generating AB-CBAs for for a wide range of important analytes.

Keywords Direct screening, Aptamer, Structure-switching, Signaling capability, Cytometric Bead Assays, Adenosine Triphosphate For many decades, people have been devoted to develop sensitive and robust analytic biochemistry assays for quantitative determination of important analytes in liquid or wet samples for research and diagnostic applications, such as bioassays, ELISA, RPA and PCR. 1 Recently, cytometric bead assays have gained intensive interests because of their multiple significant advantages, including (1) capability of multiplex evaluation of various analytes in a single sample; (2) rapid sample measurements of many replicas in a single platform; (3) need of small sample volumes for gathering data; (4) reproducible and comparative results; (5) direct comparison with other assays. 2 Particles are generally employed as solid supports in cytometric bead assays and flow cytometers are generally used for analyzing the particles. 1 It has been regarded as an excellent alternative to enzyme-linked immunosorbent assays (ELISAs) for efficient and simultaneous measurement of different panels of analytes. The application of cytometric bead assays to interesting analytes other than oligonucleotide generally requires immobilization of specific molecular recognition elements on beads. The most commonly used molecular recognition elements is monoclonal antibodies (mAbs). However immobilized mAbs are generally at the risk of denaturation that in turn results in failure of the assay. 2


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Aptamers are single-stranded (ss) oligonucleictides that have specific affinity to targets. 3 They potentially offer many advantages over mAbs: e.g. they are chemically rather than biologically synthesized, thermo-stable, undergo reversible folding, and 10 to 100 fold less expensive to manufacture than mAbs. 3 Among them, signaling aptamers are aptamer probes that are capable of generating detectable signals (such as fluorescent, and electrochemical signals) upon target binding and thus have been widely used for constructing biosensors. 4–11 Specifically, signaling aptamers have been coupled to the surface of beads, 8,12–14 enabling cytometric bead assays for viruses, 15,16 bacterials, 17 proteins, 18,19 nucleic acids, 20,21 small molecules, 13,22 and metal ions 23–25 (i.e. aptamer based cytometric bead assays, denoted as ‘AB-CBAs’). However, up to date, most of the aptamers are generated in free state in solution, and therefore the incorporation of these existing aptamers to the bead surface may alter the folding conformations and lower or even lose the performance of the aptamers. Aptamers that are capable of signaling at the bead surface, especially those that are applicable to AB-CBAs are still rarely reported. Toward this end, in this paper we propose to directly screen for AB-CBAs under the practical analyzing conditions and report the direct screening strategy for AB-CBAs with adenosine triphosphate (ATP) as a model target. ATP is a small molecule that widely exist in cells as a coenzyme and plays key roles in chemical energy transportation within cells for metabolism. 26,27 Moreover, ATP is also regarded as an indicator for cell functions such as viability and injury. 28 Thus ATP detection has proven to be of great biological importance. 13,29,30 In details, we transformed the ss DNA library to a monoclonal library of (∼ 108 ) aptamer particles through emulsion PCR as described in, 31 with each aptamer particle displaying ∼ 105 identical aptamers. The aptamer particles were then quantitatively screened via flow cytometry and only those with the highest fluorescent-signal change were isolated for subsequent PCR amplification. The monoclonal aptamer particles generated from the final screening pool through colony PCR could be directly employed as AB-CBAs for the target. This screening strategy features high efficiency: we obtained high-performance AB-CBAs for

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ATP within only three rounds of screening and we report one new ATP aptamer sequence capable of strong signaling at bead surface. More importantly, the direct screening feature of this strategy via flow cytometry assures the performance of the generated AB-CBAs for practical detection and diagnosis applications. Although the particle-display screening approach is not newly developed in this paper, 25,32,33 here we report a novel application of this method for isolation of structure-switching signaling aptamers at bead surface and even direct generation of AB-CBAs for a broad spectrum of target analytes of high interests.

Results and discussion Library design Our AB-CBA library features a constant domain (Fig. 1A, red), flanked by two random domains (Fig. 1A, blue). The random domains are further flanked by forward and reverse primer-binding domains for PCR (Fig. 1A, purple) with poly-T sequences inserted between them to increase the flexibility. We employed two fluorescence reporters to quantitatively distinguish aptamers that would switch their structure upon binding to the target from those that would not. 6 The ‘red reporter’ was labeled with Cy5 and hybridized to the constant domain of the library (Fig. 1A, red). This reporter served as an indicator of sequence structure and reported whether it was in the folded or linear state; when the aptamer exhibited high target affinity and underwent a large-scale structure change, the red reporter would be dehybridized from the constant domain, causing a decrease in red fluorescence (Fig. 1B). For sequences with no notable target affinity or aptamers with no signaling ability, the red reporter would remain hybridized, resulting in stable red fluorescence. The second ‘green reporter’ was labeled with FAM, and hybridized to the reverse PCR primer-binding domain (Fig. 1A, green). This reporter hybridized to all sequences regardless of their structural state, and served as a means of calibrating the number of sequences being displayed on particles. 4


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Figure 1: (A) Design of the AB-CBA library. Each library sequence features a fixed sequence at the center between two random sequences, further flanked by PCR forward and reverse primer-binding sites. The fixed sequence is hybridized to a Cy5 labeled antisense oligonucleotide (‘red reporter’), which reveals structure-switching events, and the reverse primer site is hybridized to a FAM-labeled oligonucleotide (‘green reporter’) that labels all aptamer-displaying particles. (B) True signaling aptamers on the particles will undergo a conformational change induced by target-binding that leads to the release of the red reporter and hence the loss of red fluorescence of the aptamer particle. This change of red fluorescence is readily detectable by FACS.

Overview of AB-CBA screening For the AB-CBA screening, firstly we converted aptamer library in solution phase into monoclonal aptamer particles using emulsion PCR (Fig. 2, steps 1 & 2). This technique uses water-in-oil emulsions to ensure that there are typically no more than one DNA template, one FP coated magnetic bead, as well as PCR reagents in each droplet (See Methods for details). We designed the process such that each of the resulting aptamer particles displays ∼ 2 × 105 copies of identical aptamer sequences on their surface. Next, we hybridized the red and green reporters to our aptamer particle library (step 3). Ideally, each aptamer particle should exhibit strong green and red fluorescence. Next we added our target molecule (step 4). Aptamer particles that display aptamers with low target affinity will continue to exhibit high green and red fluorescence, because both reporters would remain hybridized to the aptamer. On the other hand, aptamer particles with signaling capability will lose their

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red fluorescence while maintaining green fluorescence, as depicted in Fig. 1B. A two-color fluorescence-activated cell sorting (FACS) device was employed for the separation of these two populations (‘binding-screen’). The sort gate was defined so that the aptamer particles showing high green and low red fluorescence was isolated (Fig. 2 step 5, see inset). We then applied a two-round screening strategy as reported in the ref. 25 because low red fluorescence could arise from other scenarios. For example, some particles will display no aptamers at all, and thus exhibit no red fluorescence; these particles can be readily distinguished and eliminated based on the simultaneous absence of signal from the green reporter. Importantly, certain sequences can undergo self-hybridization, resulting in dehybridization of the red reporter even when no target was at present. Thus a ‘folding-screen’ was performed after the binding-screen for distinguishing the true signaling aptamers from these self-hybridizing aptamers. In this process, we directly re-incubate the aptamer particles from the binding-screen with both reporters, and performed a second two-color FACS screen in the absence of target, discarding aptamer particles that exhibit low red fluorescence (Fig. 2 step 6 and 7, see inset). This population of aptamer particles was presumably incapable of hybridizing to the red reporter due to an innate tendency towards self-hybridization. Finally, we amplified the aptamer particles collected from the folding-screen via PCR and generated an enriched aptamer pool for the next screening round or for colony PCR for generation of CBA aptamer particles (Fig. 2 step 8 and 9).

AB-CBA screening for ATP We generated novel CBA aptamer particles for a very common and important biological molecule ATP starting with an initial random particle library of 108 aptamer particles (see Fig. 3A). In the absence of target (ATP), 8.0% of the aptamer particles hybridized properly with green and red reporters (Fig. 3A, row 1, column 1) and a large portion of the particles did not display any aptamers as shown by the low green fluorescence. This was a result of Poisson statistics to ensure that no more than one template was present during the emulsion 6


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Figure 2: Overview of AB-CBA screening strategy. Aptamer particles are synthesized from a nucleic acid library through emulsion PCR (step 1-2). The aptamer particles are then hybridized with red and green reporters (step 3) and incubated with target (step 4). Those aptamer particles with strong signaling capability are sorted by FACS based on loss of red fluorescence (step 5). The isolated aptamer particles are re-hybridized with red and green reporters (step 6) and screened again in the absence of target to eliminate self-hybridizing sequences (step 7). The isolated aptamer particles are amplified through PCR for the generation of an enriched aptamer pool (step 8) that can either be used for monoclonal particle based CBAs through colony PCR (step 9) or used to synthesize aptamer particles for additional screening (step 1).

PCR process 32 to generate monoclonal aptamer particles. Importantly, we observe that ∼ 0.5% of the aptamer particles exhibited low red fluorescence in the absence of the target, indicating self-hybridized aptamers. In the first screening round (R1), we challenged the aptamer particle library with 8 mM ATP and observed that the population of high-green, low-red aptamer particles increased to 1.0% from 0.5% (Fig. 3A, row 1, column 2). This population contains true target-responsive aptamers in addition to the self-hybridized aptamers described above, and we therefore collected this population for the folding-screen. To eliminate self-hybridized aptamers from this population, we re-hybridized the collected aptamer particles with both the green and 7


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Figure 3: (A) AB-CBA screening for ATP aptamer particles. FACS plots show multiple screening rounds at different ATP concentrations ([T]). The left column represents the starting population, the middle column represents the first FACS screen (binding-screen) for aptamers that undergo target-induced structure-switching. The right column depicts the second FACS screen (folding-screen) to eliminate false-positives that undergo folding without the target. In each plot, the ‘sort gate’ of interest is marked in blue solid box and the ‘reference gate’ is marked in dashed box, with labels indicating the percentage of the total aptamer particle population represented. (B) Number of shifted aptamer particles from high to low red fluorescence for each round at ATP concentration of 4 mM (red bars to the left of each group) and 8 mM (blue bars to the right of each group).

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red reporters and performed the folding-screen without adding ATP (Fig. 3A, row 1, column 3). Here, we discarded aptamer particles that showed low red fluorescence (i.e. population that cannot hybridize to the red-reporter) and only collected those that exhibit high red and green fluorescence (approximately 27.5%). We PCR amplified the collected aptamer particles and used the resulting aptamers to generate aptamer particles for the second round of screening. In the second round (R2), we maintained the concentration of ATP at 8 mM, and observed that ∼ 1.6% of the population, i.e. 8.2% (Fig. 3A, row 2, column 2) minus 6.6% (Fig. 3, row 2, column 3), showed a shift in red fluorescence after incubating with target (Fig. 3A, row 2, column 1 and 2). After subjecting this pool to the folding screen, we collected approximately 50.4% of the aptamer particles that exhibit high red and green fluorescence as described above. In the third round (R3) (Fig. 3A, rows 3), we increased the stringency of the binding screen by reducing the concentration of ATP to 2 mM. Under this condition, we isolated 0.9% of the aptamer particles from R3, and in turn subjected to the folding screening. We concluded the selection after R3, because the resulting pool only showed a minimal additional decrease in red fluorescence compared with the previous pool when challenged with the same concentration of ATP. To verify the enrichment of signaling aptamer particles from R1 to R3, we counted the number of shifted aptamer particles from high to low red fluorescence at the ATP concentration of 4 mM and 8 mM (Fig. 3B). The R3 pool exhibited clear enrichment of signaling aptamer particles compared to the initial library since the number of shifted aptamer particles increased dramatically at both ATP concentrations. Although not necessary for the generation of AB-CBA for ATP, we still obtained aptamer sequences in the final R3 pool through Sanger sequencing (see Methods). Obtained aptamer sequences with minimal site mutations are listed in Fig. 4, where lower-case letters represent the forward and reverse primer binding sites. Please note that this sequence does not appear

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to share sequence homology with previously reported ATP aptamers. 6,34,35

Figure 4: Representative ATP aptamer sequences in the final R3 pool with minimal site mutations. The lower case letters represent primer binding sites. The constant domain where the red reporters bind is marked in red and the random domains are marked in blue. Among them, the first sequence showed the maximum red fluorescent signal change upon binding to the target ATP and was selected for subsequent measurements (denoted as ‘ATP-CBA’ aptamer).

Dissociation constant measurements We synthesized monoclonal aptamer particles from individual aptamers in the final R3 pool by cloning them into the pCR4-TOPO vector and transforming them into competent E. coli cells (see Methods). We then examined the binding affinity and signaling properties of monoclonal aptamer particles through a flow cytometry based assay (see Methods) for the dissociation constant of aptamer particles after hybridization with the red reporter (denoted AP AP as KD ) and identified the aptamer particle with the lowest KD value (i.e. best signaling

performance) as shown in Fig. 5A (see Fig. S1 in Supporting Information for more fluorescent graphs). The binding data was fitted using the isotherm equation for multiple ligands AP /[ATP])n ], where Fb , Fm assuming no target cooperativity: F = Fb + (Fm − Fb )/[1 + (KD

are the background and maximum fluorescence of aptamer particles respectively, while n is the number of ATP bound to each aptamer molecule. We found that for the best signaling AP aptamer particle, KD = 4.79 ± 0.09 mM and n = 4.63 ± 0.35. The aptamer particle with

this aptamer sequence (the first sequence as listed in Fig. 4, denoted as ‘ATP-CBA’) were specifically chosen for subsequent experiments. AP Judging from the KD value, the affinity of the ATP-CBA aptamer with ATP looks

weaker than that for the previously reported ATP aptamers 34,36 for the following two rea10


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AP measurement via a flow cytometry assay for the ATP-CBA aptamer parFigure 5: (A) KD ticles. Error bars are the standard deviation calculated from 10000 particle measurements on flow cytometer. The red solid line shows the fit using the binding equation for multiple ligAP ands, yielding KD = 4.79±0.09 mM. (B) KD measurement via a microscale thermophoresis assay for the ATP-CBA aptamer in free state without hybridization with the red reporter. Error bars are the standard deviation calculated from two independent measurements. The fit using the regular binding equation (the red solid line) gives KD = 26.36 ± 6.96 µM. (C) Specificity measurement of the ATP-CBA aptamer particles at 8 mM of concentration. The gray bar indicates the relative binding of ATP-CBA aptamer particles challenged by the target ATP while red, green, blue, cyan, and purple bars represent the relative binding challenged by nontargets GTP, GDP, GMP, ADP, and AMP respectively. Error bars are the standard deviation obtained from 10000 aptamer particles. 11


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sons. One is that the dissociation constant for the reported ATP aptamers were generally AP determined for simple binding of the aptamers in free state. Yet here KD was measured for

the entire ATP-CBA aptamer particles when the aptamer was immobilized to bead surface and underwent structure-switching upon binding, which might highly affect the affinity of AP the ATP-CBA aptamer to ATP. More importantly, KD for the ATP-CBA aptamers was

determined in the presence of the red reporter which acted as a competitor for the target AP ATP. So KD was actually “effective dissociation constant” after competing with the red

reporter. 25 To confirm and further understand the binding of the ATP-CBA aptamer with ATP, we used a microscale thermophoresis assay (see Methods) for the assessment of the dissociation constant of the ATP-CBA aptamer in free state and without hybridization with the red reporter (denoted as KD ), as shown in Fig. 5B. The binding data was fitted using the regular isotherm equation: F = Fb + (Fm − Fb )/(1 + KD /[ATP]), where Fb and Fm are the background and maximum fluorescence of the aptamer respectively, and we found that the dissociation constant of the ATP-CBA aptamer KD = 26.36 ± 6.96 µM, comparable with that of the reported ATP aptamers. 34,36

Specificity test for the ATP-CBA aptamer particles We then tested the specificity of the ATP-CBA aptamer particles by challenging the generated aptamer particles with nontarget molecules that have similar structure with ATP as shown in Fig. 5C. To readily compare the specificities, we calculated the relative binding of the ATP-CBA aptamer particles to the target (ATP) and non-target molecules (i.e. GTP, GDP, GMP, ADP, and AMP) as a percentage of particles maximal fluorescence change. We first determined the red fluorescence of the aptamer particles alone with and without hybridization to the red reporters (Fmax or Fmin respetively). Next we computed the maximum fluorescence change by subtracting Fmin from Fmax . Then we calculated the fluorescence change for each target or non-target molecule by subtracting the fluorescence of the particles 12


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incubated with a given molecule (Fi ) from Fmax . Since the quantity of either bound aptamers on the surface of the particle or released red reporter strands is directly proportional to the fluorescence change for each aptamer particle, the fraction of bound aptamers can be represented as (Fmax − Fi )/(Fmax − Fmin ). Based on the specificity measurement (Fig. 5C), we demonstrate that the ATP-CBA aptamer particles exhibited excellent specificity to ATP target, indicating that both the triphosphate and the adenosine moieties are involved in this specific recognition. We also noticed that the ATP-CBA aptamer particles showed higher affinity to GTP than other nontarget analogues. This suggests that triphosphate recognition attributed to the like charge attractions (i.e. bridging interactions) mediated by Mg2+ in PBSMT buffer may play more important roles in the overall affinity to ATP as discussed in the work by Sazani et al. 36

Conclusion In this work, we describe a direct screening strategy for generating aptamer based cytometric bead assays. The crux of our method lies in the transformation of nanometer-sized solutionphase aptamers into micrometer-sized aptamer particles. Subsequently the signaling activity of every aptamer particle in a particle library is measured and essentially individual aptamer particles are sorted at a high throughput (108 particles per hour) via FACS. In this manner, we are able to directly screen for AB-CBAs with strong signaling capability under practical analyzing conditions. As a demonstration, we report the generation of AB-CBAs for ATP with high affinity and specificity within only three rounds of screening. One new aptamer sequence that binds to ATP with strong signaling ability on the surface of a solid support is also reported. We believe that the rapid and direct screening features of this strategy make it an excellent platform for generating AB-CBAs for for a wide range of important analytes.

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Methods DNA preparation The initial ss DNA library was synthesized by Sangon Biotech with hand mixing and normal desalting purification conditions. The library contains 97-nucleotide (nt) in total, with a 15-nt constant domain (with TTT sequences at either side) between a pair of randomized domains (10- and 20-nt), which were in turn flanked by two 23-nt PCR primer sites. The sequence of each library member was as follows: 5’-cct ctc tat ggg cag tcg gtg at-[10N]TTT-CTG CAG CGA TTC TTG-TTT-[20N]-gga gaa tga gga acc cag tgc ag-3’ (where lower-case letters show primer binding sites). Each randomized site of the library had an equal probability (25%) of A, T, G or C bases. The sequence of the central constant domain was specially designed so that it could hybridize to a 15-nt Cy5-labeled red reporter sequence (5’-CAAGAATCGCTGCAG-Cy5-3’). During screening, we also hybridized the aptamers to a 23-nt carboxyfluorescein- (FAM-) labeled green reporter (i.e. FAM-labeled reverse primer) sequence (5’-FAM-CTG CAC TGG GTT CCT CAT TCT CC-3’) and forward primer complementary oligonucleotide (5’-ATC ACC GAC TGC CCA TAG AGA GG-3’). Unlabeled, 5’-amino-modified, FAM-modified PCR primers (green reporter) and Cy5-modified red reporter oligonucleotides were all synthesized by Sangon Biotech with HPLC purification.

Generation of forward primer coated particles We washed 500 µL of magnetic particles (DynaBeads MyOne Carboxylic Acid from Invitrogen, 1 µm in size, 107 /µL) once with 500 µL of 0.01N NaOH and then 5 times with 500 µL of nuclease-free water. The washed particles were re-suspended in a 150 µL reaction mixture containing 0.2 mM 5’-amino-modified forward primers (5’-amino-PEG18-CCT CTC TAT GGG CAG TCG GTG AT-3’), as well as 200 mM NaCl, 1 mM imidazole chloride, 250 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 50% v/v dimethyl sulfox14


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ide (DMSO). In this way 5’-amino-modified forward primers were covalently coupled to the particles. The incorporation of the PEG18 as a spacer was to minimize undesired interactions among the aptamers on the particle surface. Magnetic particles were mixed well with the above reagents through vortexing, and then sonicated and incubated overnight at room temperature on a rotator. After forward primer coupling was complete, the particle surface was blocked with amino-modified PEG12 (MA(PEG)12 from Thermo Fisher Scientific, dissolved in DMSO) through the following procedure. We incubated the particles in 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (100 mM, pH 4.7) with 250 mM EDC and 100 mM N-hydroxysuccinimide (NHS) for 30 minutes at room temperature so that the remaining carboxyl groups converted to amino-reactive NHS-ester, followed by conjugation with 20 mM MA(PEG)12 in MES buffer for one hour. The particles were washed 4 times with 500 µL of TE buffer (10 mM Tris, 0.1 mM EDTA, pH = 8.0), then resuspended in 500 µL of TE buffer and stored at 4 ◦ C for subsequent experiments.

Generation of aptamer particle library Aptamer particles were generated through emulsion PCR as described in ref. 31 All the reagents were purchased from Sangon Biotech unless otherwise specified. The oil phase was prepared by mixing 4.5% Span 80, 0.05% Triton X-100, and 0.40% Tween 80 in mineral oil. The aqueous phase was composed of 2× PCR Master Mix, 3.5 mM of each dNTP, 40 nM forward primers, 3 µM reverse primers, 0.25 U/µL of GoTaq Hot Start Polymerase (from Promega), 2 pM DNA template, 25 mM MgCl2 , and 3 × 108 /mL forward primer coated particles in nuclease-free water. We then generated water-in-oil emulsions by dropwisely adding (over 30 seconds) 1 mL of the aqueous phase to 7 mL of oil phase in a DT-20 tube (IKA) locked onto an Ultra-Turrax Device (IKA) stirring at 620 rpm. The mixture was continuously stirred for another 5 min after the aqueous phase was added. The emulsions were distributed every 100 µL in aliquots into ∼ 80 wells of a 96-well PCR plate. PCR amplification was performed under the following cycling conditions: 95 ◦ C for 3 15


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minutes, followed by 50 cycles of 93 ◦ C for 15 seconds, 59 ◦ C for 30 seconds and 72 ◦ C for 75 seconds. Subsequently the emulsions were collected into a 50 mL tube and broken by 10 mL of 2-butanol. After vortexing for 30 seconds, we centrifuged the particles at 3, 000 × g for 5 minutes and carefully removed the oil phase. The particle pellets were re-suspended in 1 mL of emulsion breaking buffer (100 mM NaCl, 10 mM Tris-HCl, 1% Triton X-100, 1 mM EDTA, pH 7.5), and then transferred to a new 1.5 mL tube. After the particles were centrifuged at 21, 000 × g for 1 minute, the supernatant was carefully removed via magnetic separation (MagJET Separation Rack, Thermo Fisher Scientific) and the particles were transferred to a new 1.5 mL tube. This washing step with emulsion breaking buffer was repeated once more and the remaining supernatant was removed. Next we washed the particles once with 100 µL of TE buffer, and re-suspended them in 100 µL of TE buffer. We generated ss DNA on the particles in the following steps. The particles were magnetically concentrated for 1 minute for the removal of supernatant. The particles were then re-suspended and incubated in 200 µL of 100 mM NaOH for 20 minutes. The supernatant was carefully removed via magnetic separation again. We finally washed the particles twice with 100 µL of TE buffer, and re-suspended them in 100 µL of TE buffer.

AB-CBA screening Prior to the screen, aptamer particles were pretreated in the following steps. We first hybridized them with 2 µM each of green and red reporters, as well as forward primer complementary oligonucleotide in PBSMT buffer (DPBS with additional 2.5 mM MgCl2 , 0.025% TWEEN-20) for 30 minutes, and then washed them 3 times with PBSMT buffer. After emulsion PCR, it is expected that approximately 80% of the aptamer particles display only forward primers on their surface according to Poisson distribution and thus show negligible binding to targets. The ‘reference gate’ to calibrate non-specific binding for particle display screen can be well established according to the negative control determined by these forward primer only particles. During each round of screening, we incubated ∼ 108 aptamer parti16


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cles in 2 mL of PBSMT in the presence of ATP for 20 minutes at room temperature. The application of two-color labelling (with green and red reporters) enabled the monitoring of the fraction of particles that displayed aptamers, low-affinity or self-hybridizing sequences in the aptamer pool respectively via a flow cytometer (BD FACSAria). Essentially the fluorescence distribution of aptamer particles at different target concentrations from 1 mM to 16 mM was monitored and the target concentration was specifically chosen for sorting so that 0.3% to 2% of aptamer particles showed a red fluorescence reduction. The aptamer particles displaying low red and high green fluorescence were collected and concentrated via magnetic separation. They were re-hybridized with 2 µM of each green and red reporters, as well as forward primer complement in PBSMT buffer. We washed these aptamer particles 3 times with PBSMT buffer and sorted again via FACS in the absence of target for those displaying high red and green fluorescence. The ultimately isolated aptamer particles were PCR amplified for the generation of an enriched pool for aptamer particle synthesis in the next screening round.

Cloning and sequencing We cloned the aptamer pool from the final ATP screening round (R3) using a TOPO TA cloning kit (Life Technologies). The DNA in the final pool after PCR amplification was directly inserted into a plasmid vector. We transformed the vectors into chemically competent TOP10 E. coli cells grown on pre-poured agar plates with 50 µg/mL kanamycin at 37 ◦ C overnight. We carefully picked individual colonies and transferred them to new agar plates with kanamycin by pipette tip touching. The cells from individual colonies were grown at 37 ◦ C overnight and were used for subsequent monoclonal aptamer particle synthesis. The plates were sent for Sanger sequencing by Sangon Biotech.

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Monoclonal aptamer particle synthesis (Colony PCR) Monoclonal aptamer particles (aptamer particles displaying each unique aptamer sequence) were synthesized directly from the colonies generated in the previous subsection through asymmetric colony PCR. Each 50 µL reaction solution contained 2× PCR Master Mix (Thermo Fisher Scientific), 100 nM forward primer and 1 µM unlabelled reverse primer. We added template by carefully touching a pipette tip on the designated colony and transferring the colony to the 50 µL PCR mixture. Colony PCR conditions were as follows: 95 ◦ C for 2 minutes, then 20 cycles of 95 ◦ C for 15 seconds, 59 ◦ C for 30 seconds, and 72 ◦ C for 45 seconds, and a final extension at 72 ◦ C for 7 minutes. Monoclonal aptamer particles were then synthesized through particle PCR using the asymmetric PCR product for template. Each 60 µL particle PCR contained 2× PCR Master Mix (Thermo Fisher Scientific), 1 µM FAM-modified reverse primer, 20 mM MgCl2 , 3 µL of asymmetric PCR product template and 3 × 107 forward primer coated particles. PCR was performed under the following conditions: 95 ◦ C for 2 minutes, then 24 to 32 cycles of 95 ◦ C for 15 seconds, 59 ◦ C for 30 seconds, and 72 ◦ C for 75 seconds, and a final extension step at 72 ◦ C for 7 minutes in the end. To avoid aggregation of particles during PCR process, we vortexed and sonicated the reagent tubes every four cycles in the middle of the 72 ◦ C extension step. After the particle PCR, we checked PCR efficiency based on the FAM fluorescence intensity of the aptamer particles as measured by flow cytometry and adjusted particle PCR cycle numbers accordingly. Finally, ss aptamers were generated by incubation with 200 µL of 100 mM NaOH for 20 minutes, and then washed with 100 µL of TE buffer twice.

AP measurements via flow cytometry KD AP The KD values of all sequences were determined by a flow cytometry based binding assay.

Firstly we hybridized aptamer particles with each of 2 µM green reporter, red reporter and forward primer complement respectively in PBSMT buffer. The excess reporters and for18


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ward primer complement were removed by washing three times with PBSMT buffer. We then incubated 1.5 µL aliquots of pretreated aptamer particles in 50 µL PBSMT buffer with titrations of ATP ranging from 1 mM to 16 mM at room temperature for 20 minutes on a rotator. Finally we measured Cy5 fluorescence using an Accuri C6 flow cytometer (BD Biosciences).

KD measurements via microscale thermophoresis The KD value of the ATP-CBA aptamer in free state and without hybridization to the red reporter was determined using a microscale thermophoresis based assay for molecular interactions. The ATP-CBA aptamer was synthesized with a Cy5 modification at the 5’ end. The aptamer at 5 nM (without the presence of the red reporter, the green reporter, or forward primer complement) was incubated with a two-fold titration of ATP ranging from 21.36 nM to 700 µM concentrations in PBSMT buffer. The binding of the ATP-CBA aptamer with ATP was analyzed by a Monolith Pico device (NanoTemper Technologies) at room temperature. The KD of the ATP-CBA aptamer was determined based on the fluorescence shift due to normal thermophoresis.

Acknowledgement This study was supported by the National Key R&D Program of China (2017YFC1600603), the National Natural Science Foundation of China (21705031), the Natural Science Foundation of Anhui Province (1808085QB39, 1708085QC66), and the Fundamental Research Funds for the Central Universities (JZ2017HGTB0195, JZ2018HGTA0212). We thank Prof. Xian-Zhu Yang, and Prof. Hai-Sheng Qian for assistance with the flow cytometer.

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Supporting Information Available The following files are available free of charge. • Supporting Information.docx: Red fluorescence graphs for the ATP-CBA aptamer particles challenged by various concentrations of ATP

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Graphical TOC Entry

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Direct Screening for Cytometric Bead Assays for Adenosine

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