Identification and characterization of DNA aptamers specific for

Oct 2, 2018 - Tau proteins are proteins that stabilize microtubules, but their hyperphosphorylation can result in the formation of protein aggregates ...
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Identification and characterization of DNA aptamers specific for phosphorylation epitopes of Tau protein I-Ting Teng, Xiaowei Li, Hamad Ahmad Yadikar, Zhihui Yang, Long Li, Yifan Lyu, Xiaoshu Pan, Kevin K Wang, and Weihong Tan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08645 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018

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Identification and characterization of DNA aptamers specific for

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phosphorylation epitopes of Tau protein

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I-Ting Teng1,§, Xiaowei Li1,§, Hamad Ahmad Yadikar#, Zhihui Yang#, Long Li$, Yifan

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Lyu$,&, Xiaoshu Pan$, Kevin K. Wang #%* and Weihong Tan§,&*

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§

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for Research at Bio/Nano Interface, UF Health Cancer Center, UF Genetics Institute and

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McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA

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&

Department of Chemistry, Department of Physiology and Functional Genomics, Center

Molecular Science and Biomedicine Laboratory, State Key Laboratory for Chemo/Bio-

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Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of

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Life Sciences, and Aptamer Engineering Center of Hunan Province, Hunan University,

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Changsha 410082, China

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#Department of Emergency Medicine, Department of Chemistry, Department of

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Neuroscience, Department of Psychiatry, McKnight Brain Institute, University of Florida,

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Gainesville, Florida 32611, USA

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%

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Medical Center, 1601 SW Archer Rd., Gainesville FL 32608, USA

Brain Rehabilitation Research Center (BRRC), Malcom Randall Veterans Affairs

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*Correspondence should be addressed to

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Weihong Tan ([email protected]) or Kevin Wang ([email protected]).

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1: Equal contribution

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Abstract Tau proteins are proteins that stabilize microtubules, but their

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hyperphosphorylation can result in the formation of protein aggregates and, over

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time, neurodegeneration. This phenomenon, termed tauopathy, is pathologically

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involved in several neurodegenerative disorders. DNA aptamers are single-

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stranded oligonucleotides capable of specific binding to target molecules. Using

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tau epitopes predisposed for phosphorylation, we identified 6 distinct aptamers

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that bind to tau at two phosphorylatable epitopes (Thr-231 and Ser-202) and to

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full-length Tau441 proteins with nanomolar affinity. In addition, several of these

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aptamers also inhibit tau phosphorylation (IT4, IT5, IT6) and tau oligomerization

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(IT3, IT4, IT5, IT6). This is the first report to identify tau epitope-specific

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aptamers. Such tau aptamers can be used to detect tau in biofluids and uncover

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the mechanism of tauopathy. They can be further developed into novel

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therapeutic agents in mitigating tauopathy-associated neurodegenerative

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disorders.

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INTRODUCTION Tau proteins are microtubule-associated proteins known to promote the

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assembly of microtubules and to maintain microtubule integrity, which is essential

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for axonal transport and morphogenesis.1, 2 Normal tau proteins bind with

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microtubules and prevent these track-like structures from breaking apart,

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allowing nutrients and molecules to be transported along the cells. However, tau

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is also found to be pathologically involved in several neurological disorders,

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termed tauopathies, in which aggregations of tau are deposited in brain

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neurons.3 In the case of Alzheimer’s disease, pathological tau proteins self-

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assemble into paired helical filaments, which later aggregate into insoluble

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neurofibrillary tangles.4 The transport system for neurons is disrupted along the

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process, causing nutrients and other essential supplies to cease moving along

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the cells. Neurons with tangles and non-functioning microtubules consequently

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undergo apoptosis and eventually cell death. Such phenomenon is also observed

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in a range of other neurodegenerative diseases.5, 6 Various forms of insoluble

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abnormal tau aggregates are involved in tauopathies, but they share a common

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composition of hyperphosphorylated tau.

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Aptamers are nucleic acid probes capable of specific binding to defined

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targets.7 They are selected through an amplification-evolution process termed

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systematic evolution of ligands by exponential enrichment (SELEX).8 Aptamers

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have been selected against a variety of targets, including metal ions,9 fluorescent

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dyes,10 amino acids,11 nucleotides,12 antibiotics,13 metabolites,14 peptides,15

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proteins,16 viruses,17 organelles,18 or even whole cells.19 As such, aptamers have

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shown remarkable specificity in discriminating targets from their analog

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counterparts, such as differentiating among homologous proteins differing by

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only a few amino acids20 or one single amino acid21, or even between

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enantiomers.22 Since molecular recognition is essential in many biological

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processes, aptamers can potentially inhibit the functions of their targets. For

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instance, binding of thrombin with its aptamers has been shown to undermine its

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activity and decrease the rate of blood clotting.23

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The most fundamental difference between healthy tau and pathological tau can be ascribed to the level of phosphorylation. Abnormally

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phosphorylated tau lesion causes death of neuron cells, resulting in irreversible

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and progressive neurodegeneration. Understanding the origin and mechanism of

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tauopathy is a key step toward developing a means to delay or even fight against

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it. Since pathological tau loses its affinity for microtubules due to an abnormally

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high degree of phosphorylation, we hypothesized that aptamers binding to

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phosphorylatable regions on tau protein might be useful in studying the molecular

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mechanism(s) underlying tauopathy. It was also anticipated that aptamers

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binding to the phosphorylated sites on tau could be exploited to investigate the

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formation of hyperphosphorylated tau aggregates and detect the phosphorylation

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level. Therefore, we herein describe a SELEX process using fragments of

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phosphorylatable regions from tau protein and their corresponding

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phosphorylated forms as targets to search for site-specific tau aptamers for

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potential further use as tauopathy-detecting agents and possible therapeutic

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agents.

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RESULTS AND DISCUSSION

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Tau epitope-specific aptamer discovery and selection

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The selection began with a library containing 20 nmol of primer-flanked,

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66-nucleotide-long, single-stranded DNA. Four phosphorylatable peptide

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epitopes from tau and their corresponding phosphorylated peptides (Table S1)

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were used as putative targets (Figure 1). The detailed screening strategy is

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described in the Supplementary Information.

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Figure 1. Tau aptamer discovery by SELEX and characterization workflow. Four

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peptide epitopes from tau and their corresponding phosphorylated peptides were

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used as putative targets. The enrichment was monitored using flow cytometry.

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The most abundant 10 candidates were characterized after deep sequencing.

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The binding affinity/specificity and the inhibitory effects with the identified

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aptamers were then verified.

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The 10 most abundant sequences found in Pool #17 and their population

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percentage in each of the sequenced pools are listed in Table S2. They are

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denoted as IT1 through IT10 and were chemically synthesized and labeled with

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FAM at the 5’-end, followed by HPLC purification. Primary binding analysis was

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examined by flow cytometry. Seven out of the 10 candidates are confirmed

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aptamers (Figure 2F, K-P). Five of them (IT1, IT4, IT5, IT6, and IT9) possess

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strong and specific binding to T231 peptide, while IT3 bypassed the

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phosphorylated site and bound to both T231 and T231P peptides. Aptamer IT2,

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on the other hand, recognized not only both T231 and T231P, but it also bound

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to S202. It is noteworthy that IT8, IT9, and IT10 bore a high level of sequence

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homology to IT3, IT5, and IT2, respectively. In fact, the discrepancy only occurs

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at the 19th nucleotide (Table S3). IT9 and its predecessor IT5 displayed similar

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binding strength towards T231, suggesting that the nucleotide at position 19 for

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these two similar sequences had no significant impact on their binding abilities to

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T231. However, IT8 and IT10 both lost their binding abilities to the prospective

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targets found with IT3 and IT2, proving that the binding strength of an aptamer

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could be greatly compromised by merely altering one nucleotide within the crucial

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binding region. Based on the fact that IT8 and IT10 manifested no binding

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preference to either of the peptides, they are likely the byproducts that resulted

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from PCR amplification with edge effect.

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The initial binding tests were carried out at 4 °C to ensure having the

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optimal secondary structures for aptamers. The binding abilities of the selected

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aptamers were then examined at room temperature and at 37 °C. None of the

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aptamers lost binding ability at room temperature or 37 °C (data not shown),

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suggesting that these aptamers are suitable for future in vivo studies.

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Figure 2. Primary binding analysis between aptamer candidates and their

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putative peptide targets. (A-E) Representative predicted secondary structures of

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aptamer IT1 and its subsequent truncated forms, IT1a, IT1b, IT1c, and IT1d. (F

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and M-P) Full-length sequences IT1, IT4, IT5, IT6, and IT9 are aptamers that

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specifically bind to T231 peptide. (F-J) The binding abilities of truncated

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aptamers IT1a, IT1b, and IT1c to T231 epitope are not seriously compromised

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when compared to the full-length aptamer IT1. However, binding strength is lost

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when further truncating the stem of IT1c into IT1d. (K) Sequence IT2

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demonstrates a unique binding profile on three of the peptides (T231, T231P,

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and S202). (L) Sequence IT3 bypasses the phosphorylated site and recognizes

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both T231 and T231P.

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Sequence truncation of the selected aptamers

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The aptamers identified are full-length sequences evolved from the initial

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library, which contains the fixed primer binding regions on both ends to serve the

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PCR amplification process. However, some full-length aptamers can be

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shortened into a minimally functional sequence without compromising direct

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interaction with the target.24 Herein, we synthesized such truncated versions of

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aptamers IT1, IT2, IT3, IT4, IT5, and IT6 based on secondary structures

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predicted by Integrated DNA Technologies OligoAnalyzer Tool (Figure 2A-J,

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Table S4). Two to three T-bases were also added as a spacer next to fluorescein

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if the truncated sequence ended with G-base or G-C pair in the stem.

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We successfully identified the binding motif from aptamer IT1 as sequence

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IT1c, which is less than half the length of its original IT1 sequence. IT2 was at

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first truncated to IT2a based on an evident stem-loop motif observed in the

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predicted secondary structure and IT2a demonstrated binding ability equal to IT2

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for targets T231, T231P, and S202. Still, an even more stringent truncation of

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IT2, candidate IT2b, failed to maintain the properties of the original aptamer. A

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less exacting approach was then implemented to further shorten aptamer IT2a.

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Sequence IT2c presented binding ability similar to IT2a, but a gradual loss of

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binding ability was observed by further truncating the stem of IT2c into IT2d and

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IT2e, suggesting the importance of a stable stem for binding of aptamer IT2c to

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its targets. A less restrictive truncation of aptamer IT3 into sequence IT3c

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resulted in partial binding to T231, but it caused a complete loss of binding to 8

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T231P. For IT4 and IT5, the complete sequences are required for target

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recognition. Finally, a branched hairpin structure, IT6a, retained selective binding

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ability to T231 as aptamer IT6.

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Determination of binding kinetics/affinities of the selected aptamers

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In this kinetics study, his-tag peptides, as well as his-tag Tau441 protein,

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were immobilized on the surface of the biosensor, while aptamer candidates

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were the analytes in the solution phase. The binding interaction of analyte to

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immobilized ligand was measured in real time as the change in the number of

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molecules bound to the biosensor that caused a shift in the interference pattern

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reflected from sensor surfaces. The kinetic on-rates (kon), off-rates (koff), and

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equilibrium dissociation constants (Kd) measured for all aptamer-target pairs are

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summarized in Table S5.

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All aptamers reported here displayed high binding affinities toward Tau441

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with Kd values ranging from 5.5 nM to 68 nM. The observed on-rates (kon)

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ranged between 104 M-1s-1 and ~ 106 M-1s-1. Aptamer IT1 presented the fastest

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on-rate ((9.3 ± 1.9) ×105 M-1s-1) toward Tau441 protein, while the slowest on-rate

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((1.067 ± 0.018) ×104 M-1s-1) was detected for IT2 binding to Tau441. However,

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IT2 also demonstrated an extremely slow off-rate (koff) ((5.9 ± 1.2) ×10-5 s-1) for

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Tau441, exhibiting the lowest Kd (5.5 ± 1.1 nM) for Tau441 protein among all

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aptamers. The lowest Kd toward peptides, on the other hand, was observed

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between IT5 and T231 (5.0 ± 0.3 nM). Meanwhile, IT5 also exhibited the second

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lowest Kd (7.6 ± 0.6 nM) for Tau441 protein among all aptamers, indicating the

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high binding affinities of IT5 toward both T231 peptide and Tau441 protein.

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Although aptamer IT9 differs by only one base from IT5, as shown in Table S3, it

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showed appreciably slower kon and, therefore, higher equilibrium dissociation

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constants compared to IT5.

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While IT1 showed similar binding kinetics toward T231 and Tau441, the

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truncated aptamer IT1c exhibited a much slower off-rate for Tau441 protein than

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for T231 peptide. On the other hand, truncation of IT2 did not affect the

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association of the aptamers to T231 peptide, but the shorter aptamer IT2a did

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display a faster dissociation rate for T231 compared to IT2. In fact, the

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dissociation rates with any of the targets were increased with IT2a compared to

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the off-rates with IT2. In addition, a faster on-rate for T231P peptide was found

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with either IT2 or IT2a in comparison to that of T231. With IT2a, especially, the

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on-rate toward T231P was almost 5 times faster than its on-rate toward T231,

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and this on-rate was also 2.5-fold faster than the association of IT2 to T231P.

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However, the association between IT2a and S202 peptide showed an opposite

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tendency. The on-rate of IT2a for S202 was found to be more than 6 times

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slower than its IT2 counterpart. Therefore, the truncation of IT2 appears to

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benefit its recognition of T231P, while, at the same time, losing its binding affinity

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toward the S202 site. Moreover, while the association of IT2a to Tau441 is

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almost 50 times faster than IT2, its off-rate is also 60 times faster than that of IT2.

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Overall, the binding affinity of IT2a was weakened by the truncation. Unlike IT1c

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and IT2a, the shorter version of IT6 did not affect the binding kinetics and affinity.

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IT6a still behaved much like IT6. Finally, the on-rates of IT3 to both T231 and

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T231P peptides were found to be quite similar, but the off-rate with T231P was

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almost 2-fold faster than that with T231.

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Specificity of the selected tau aptamers against Tau441 protein

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The binding specificity of the selected tau aptamers to full-length Tau441

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protein was confirmed by nondenaturing gel electrophoresis after incubating

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each of the FITC-labeled aptamers with either target Tau441 protein or the

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individual nontarget proteins separately. Each of the tau aptamers (IT1- IT6 and

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IT9) alone displayed one main band at the lower part of the gel (Figure 3, lane 1).

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Some minor upper bands observed could have resulted from dimeric or

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multimeric forms of the oligonucleotides. The monomeric aptamers disappeared

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in the presence of the target Tau441 protein due to the formation of aptamer-

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Tau441 complexes (Figure 3, lane 2). No cross-reactivity was observed between

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the aptamers and the nontarget proteins, including S100B (S100 calcium-binding

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protein B) (11 kDa), UCH-L1 (Ubiquitin carboxyl-terminal hydrolase L1) (25 kDa),

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α casein (23 kDa), β casein (24 kDa), BSA (bovine serum albumin) (66 kDa), and

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IgG (immunoglobin G) (150kDa) (Figure 3, lane 3-8). In particular, S100B25 and

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UCH-L126 are important references because they are also brain-associated

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protein and traumatic brain injury (TBI) biofluid biomarker proteins.27

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Figure 3. Binding specificity of the tau aptamers towards tau protein. Gel

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electrophoresis of each FITC-labeled tau aptamer, (A) IT1, (B) IT2, (C) IT3, (D)

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IT4, (E) IT5, (F) IT6, and (G) IT9, after incubation with the protein of interest.

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Aptamers form binding complexes with tau and therefore show a smaller

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migration in the presence of tau. S100B, UCH-L1, α casein, β casein, BSA, and

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IgG are nontarget reference proteins and have no retention effect on the

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migration of aptamers under electrophoresis.

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Aptamer-based sandwich ELISA for detection of Tau

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To further study the feasibility of using tau aptamers for tau protein

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detection, a sandwich enzyme-linked immunosorbent-assay (ELISA) was

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performed in triplicate. Tau protein was first captured by DAKO antibody. The

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biotin-labeled IT4 aptamer was used as the detection probe to specifically

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recognize tau protein. The 620-nm absorbance was recorded after incubating

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samples with streptavidin-labeled polyHRP enzyme and TMB substrate. As

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shown in Figure 4, IT4 aptamer strongly binds to tau protein with a dose-

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dependent increase in signal, as opposed to nontarget phosphorylated tau,

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indicating the possibility of utilizing IT4 aptamer to specifically quantify the tau

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level in biological samples.

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Figure 4. Aptamer-antibody sandwich ELISA for detection of tau. Both tau and

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phosphorylated tau can be captured by total tau protein. However, aptamer IT4

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only detects the presence of nonphosphorylated T231 residue. The scrambled

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random sequence (RS) and phosphorylated tau protein are included in this test

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as negative controls. All the sequences are labeled with biotin, which later reacts

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with streptavidin-labeled polyHRP enzyme and TMB substrate to reveal the

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colorimetric reaction products.

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Inhibitory effects of tau aptamers on tau phosphorylation

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To analyze the ability of tau aptamers to inhibit tau phosphorylation, tau

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was phosphorylated by kinase glycogen synthase kinase-3β (GSK3β) for 24h at

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37 oC in the presence or absence of tau aptamers. We used phospho-T231 tau

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monoclonal antibody (RZ3) to probe the phosphorylated tau. A faint 68K band

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was observed with control Tau441 protein (Figure 5A, lane 1), while an intense

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band showing a molecular shift from 68K to 70K was detected with the

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commercially available positive control GSK3β pre-phosphorylated Tau441

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(Figure 5A, lane 2). Lane 3 shows the experimentally GSK3β-phosphorylated

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Tau441 at both the monomeric 70K band and oligomeric 150K p-tau band. The

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presence of random oligonucleotide had no effect on the levels of

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phosphorylated monomeric tau or oligomeric p-tau (Figure 5, lane 4). IT1 and

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IT1c showed minor reduction of monomeric p-tau, but they had no effect on

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oligomeric p-tau (Figure 5, lane 5-6). The presence of IT2, IT2a and IT3

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somehow promoted phosphorylated tau towards the oligomeric form (Figure 5,

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lane 7-9). The reason behind this result is not fully understood yet. But we

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suspect this is due to the fact that these aptamers bind to more than one epitope

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(Figure 2K-L). Among all aptamers, IT4 eliminated both monomeric p-tau and

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oligomeric p-tau most dramatically (Figure 5, lane 10). IT5 also showed some

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inhibition on monomeric p-tau, but IT6 and IT6a had no significant effects (Figure

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5, lane 11-13).

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Figure 5. Inhibitory effects of Tau-binding aptamers on phosphorylation of

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Tau441 in vitro. (A) In vitro tau phosphorylation and oligomerization assay was

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performed by incubating Tau441 (1 µg) with aptamers (50 µM) for 1 h, followed

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by incubation with GSK3β (200 ng) for 16 h. Samples were analyzed by SDS-

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PAGE, followed by Western blotting with phospho-tau antibody (RZ3). (B)

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Quantification of monomeric p-tau (70K) and (C) oligomeric p-tau (150K) bands

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of tau protein. The monomeric p-tau (70K) and oligomeric tau (150K) bands were

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normalized by the levels of GSK3β-phosphorylated Tau441 and were shown as

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percentage. *p