Identification and Characterization of DNA ... - ACS Publications

Subscriber access provided by UNIV OF LOUISIANA

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

1

Identification and characterization of DNA aptamers specific for

2

phosphorylation epitopes of Tau protein

3

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

4

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

5 6

§

7

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

8

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

9

&

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

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

10

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

11

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

12

Changsha 410082, China

13

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

14

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

15

Gainesville, Florida 32611, USA

16

%

17

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

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

18 19

*Correspondence should be addressed to

20

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

21

1: Equal contribution

22 23

1

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Abstract Tau proteins are proteins that stabilize microtubules, but their

3

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

4

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

5

involved in several neurodegenerative disorders. DNA aptamers are single-

6

stranded oligonucleotides capable of specific binding to target molecules. Using

7

tau epitopes predisposed for phosphorylation, we identified 6 distinct aptamers

8

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

9

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

10

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

11

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

12

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

13

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

14

therapeutic agents in mitigating tauopathy-associated neurodegenerative

15

disorders.

2


Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

assemble into paired helical filaments, which later aggregate into insoluble

11

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

12

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

13

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

14

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

15

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

16

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

17

composition of hyperphosphorylated tau.

18

Aptamers are nucleic acid probes capable of specific binding to defined

19

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

20

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

21

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

22

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

23

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

3



1

shown remarkable specificity in discriminating targets from their analog

2

counterparts, such as differentiating among homologous proteins differing by

3

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

4

enantiomers.22 Since molecular recognition is essential in many biological

5

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

6

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

7

activity and decrease the rate of blood clotting.23

8 9

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

10

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

11

and progressive neurodegeneration. Understanding the origin and mechanism of

12

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

13

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

14

high degree of phosphorylation, we hypothesized that aptamers binding to

15

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

16

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

17

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

18

formation of hyperphosphorylated tau aggregates and detect the phosphorylation

19

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

20

phosphorylatable regions from tau protein and their corresponding

21

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

22

potential further use as tauopathy-detecting agents and possible therapeutic

23

agents.

4


Page 4 of 36

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

RESULTS AND DISCUSSION

2

Tau epitope-specific aptamer discovery and selection

3

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

4

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

5

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

6

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

7

described in the Supplementary Information.

8 9

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

10

peptide epitopes from tau and their corresponding phosphorylated peptides were

11

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

12

The most abundant 10 candidates were characterized after deep sequencing.

13

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

14

aptamers were then verified.

5



1

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

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

from PCR amplification with edge effect.

21

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

22

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

23

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

6


Page 6 of 36

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

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

2

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

3 4

Figure 2. Primary binding analysis between aptamer candidates and their

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

7



1

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

2

both T231 and T231P.

3

Sequence truncation of the selected aptamers

4

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

5

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

6

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

7

shortened into a minimally functional sequence without compromising direct

8

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

9

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

10

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

11

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

12

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

13

We successfully identified the binding motif from aptamer IT1 as sequence

14

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

15

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

16

predicted secondary structure and IT2a demonstrated binding ability equal to IT2

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

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


Page 8 of 36

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

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

2

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

3

ability to T231 as aptamer IT6.

4

Determination of binding kinetics/affinities of the selected aptamers

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

summarized in Table S5.

13

All aptamers reported here displayed high binding affinities toward Tau441

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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

9



1

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

2

showed appreciably slower kon and, therefore, higher equilibrium dissociation

3

constants compared to IT5.

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

10


Page 10 of 36

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

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

2

almost 2-fold faster than that with T231.

3

Specificity of the selected tau aptamers against Tau441 protein

4

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

5

protein was confirmed by nondenaturing gel electrophoresis after incubating

6

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

7

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

8

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

9

Some minor upper bands observed could have resulted from dimeric or

10

multimeric forms of the oligonucleotides. The monomeric aptamers disappeared

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

11



1 2

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

3

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

4

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

5

Aptamers form binding complexes with tau and therefore show a smaller

6

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

7

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

8

migration of aptamers under electrophoresis.

9

Aptamer-based sandwich ELISA for detection of Tau

10

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

11

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

12


Page 12 of 36

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

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

2

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

3

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

4

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

5

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

6

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

7

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

8

level in biological samples.

9 10

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

11

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

12

only detects the presence of nonphosphorylated T231 residue. The scrambled

13

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

14

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

13



1

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

2

colorimetric reaction products.

3

Inhibitory effects of tau aptamers on tau phosphorylation

4

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

5

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

6

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

7

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

8

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

9

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

10

commercially available positive control GSK3β pre-phosphorylated Tau441

11

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

12

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

13

presence of random oligonucleotide had no effect on the levels of

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

5, lane 11-13).

14


Page 14 of 36

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

percentage. *p

Identification and Characterization of DNA ... - ACS Publications

Recommend Documents