Zhang XM et al / Acta Pharmacol Sin 2003 Jul; 24 (7): 711-714

Screening of RNA molecules inhibiting human acetylcholinesterase by virtue of systematic evolution of ligands by exponential enrichment¹

ZHANG Xing-Mei, SHAO Ning-Sheng², CHI Mu-Gen, SUN Man-Ji³

Institute of Pharmacology and Toxicology, ²Institute of Basic Research, Academy of Military Medical Sciences, Beijing 100850, China

¹ Project supported by the Natural Science Foundation of Beijing (No 7022032).

³ Correspondence to Prof SUN Man-Ji. Phn 86-10-6693-0691. Fax 86-10-6821-1656. E-mail sunmj@nic.bmi.ac.cn

Received 2002-06-13 Accepted 2003-03-06

KEY WORDS acetylcholinesterase; SELEX; aptamer

ABSTRACT

AIM: To acquire the specific RNA aptamers inhibiting human red blood cell (RBC) acetylcholinesterase (AChE). METHODS: Systematic evolution of ligands by exponential enrichment (SELEX) aptamer against human red blood cell membrane AChE was selected by microtiter plate method in vitro. The specific binding to AChE was determined by gel mobility shift analysis. Microcolorispectrophotometric method was used to measure the activity of AChE. RESULTS: The aptamers to human RBC AChE were indentified by 9 reiterative rounds. At the same concentration (2.25 µmol/L), the aptamers did not bind to the recombinant human butyrylcholinesterase (rhBChE) but specifically bound to human RBC AChE and inhibited the enzyme activity. CONCLUSION: It is an effective way to isolate the specific AChE inhibitor from the vast oligonucleotide combinatorial library by virtue of SELEX.

INTRODUCTION

Systematic evolution of ligands by exponential enrichment (SELEX)^[1] is an oligonucleotide-based combinatorial chemistry high-tech using for isolation of high-affinity high-specific ligands (aptamer) for a wide variety of macromolecules and low molecular weight targets^[2,3]. Specific single-strand oligonucleotides (either RNA or DNA) with high affinity for targets are isolated from huge library of random sequence molecules by reiterative rounds of selection and amplication. Aptamers can be engineered readily to function as either therapeutics or dignostics^[4]¡. For instance, aptamers that bind to human immunodeficiency virus type I integrase or reverse transcriptase can inhibit viral replication^[5].

The present study was to acquire aptamers binding to the human acetylcholinesterase (EC 3.1.1.7, AChE) inhibiting enzyme activity. Inhibition of AChE is routinely employed for an increasing variety of clinical applications (eg, Alzheimer's disease drugs, muscle relaxants or prophylactics in protection against nerve agents attack).

MATERIALS AND METHODS

Reagents Human RBC membrane AChE ( SDS-PAGE pure, 1.1 g/L) was a gift from Dr LIAO Jian (University of Bern, Switzerland). Microtiter plate was obtained from Nunc Co (Denmark). T7 transcription kit and reverse tanscription kit were purchased from Promega (USA). PCR amplification kit was from TakaRa Co (Dalian, China). Single-stranded DNA primers and templates were synthesized by Bioasia Inc (Shanghai, China). [-³²P]UTP was from Yahui Inc (Beijing, China). All other chemicals were of AR grade.

Oligonucleotide library A population of 1×10¹⁴ DNA templates (G library) each with a 25-nucleotide (25 N) variable region and 5' and 3' fixed regions were created by oligonucleotide synthesis, and a 85-nucleotide double-stranded PCR product of each was constructed.

5' primer containing the T7 promotor underlined:

5'-TAATACGACTCACTATAGCAATGGTACGGTACT-TCC-3';

3'primer:

5'-TTAGCAAAGTAGCGTGCACTTTTG-3'.

ssDNA-G template:

5'-TAATACGACTCACTATAGCAATGGTACGGTACT

TCC(25N)CAAAAGTGCACGCTACTTTGCTAA-3';

In vitro selection The RNA pool was transcribed from the amplified DNA template using a T7 in vitro transcription kit^[6]. After purification on an 8 % denaturing polyacrylamide gel, the RNA pellet was dissolved in sodium phosphate-buffered saline (PBS, pH 7.4).

For selection of aptamers directed to AChE, 9 rounds of enrichement were carried out consecutively^[7]. For each SELEX round, microtiter plates were kept at 4 ºC overnight with AChE solution in PBS (Tab 1). RNA input into each round was also described in Tab 1. After discarding the supernatant, wells were washed with the SHMCK blocking buffer (Hepes 20 mmol/L, pH 7.35, NaCl 120 mmol/L, KCl 5 mmol/L, CaCl₂ 1 mmol/L, MgCl₂ 1 mmol/L, and casein 1 g/L) for 6 times. RNA was diluted into 200 µL of binding buffer (SHMCK blocking buffer containing 0.05 % Tween-20), denatured at 95 ºC for 5 min, annealled on ice for 5 min, then added into the AChE-coated wells and incubated at 37 ºC for 2 h. Following casting off the supernatant and washing with the binding buffer for 6 times, the dry well was placed on the top of a 95 ºC-heat block for 5 min. Standard reverse transcriptase reaction (50 µL) were performed at 48 ºC directly in the wells, and the reaction products was used for standard PCR and transcription reaction. The 5' and 3' primers were used for template amplication and reverse transcription^[6].

Mobility shift assay The binding ability of RNA to AChE was detected by gel mobility shift assay. Radiolabeled RNA samples were acquired by transcription, then heated to 95 ºC for 5 min and cooled on ice over 5 min. RNA (1.2 nmol/L) were mixed with AChE in 20 µL total volume of 1×shift buffer (Hepes 10 mmol/L, pH 7.4, KCl 100 mmol/L, dithiothreitol 1 mmol/L, MgCl₂ 1 mmol/L, edetic acid 1 mmol/L, tRNA 50 mg/L, and 5 % glycerol). The binding reaction was carried out at 4 ºC for 30 min and then fractionated by means of 8 % non-denaturing polyacrylamide gel electrophoresis (29:1, acrylamide / bisacrylamide, 1×TBE) at 4 ºC for 3 h at 80 V. The gel was dried and exposed to autoradiographic film at -20 ºC. Mobility shifts of RNA-rhBChE were performed in the same way.

AChE assay Different concentrations of 9-round aptamers (G₉) and the primary RNA library (G₀) in PBS were separately denatured at 95 ºC for 5 min and cooled on ice over 5 min, then added AChE solution and incubated at 4 ºC for 1 h. The enzyme assay was performed as described previously^[8]. In this expriment the normal AChE activity was 5.8 mol·h^-1·g^-1 (protein). The relative AChE activities of the samples were expressed in terms of percentages of the normal enzyme control.

RESULTS

Protein (AChE) and RNA input in each round RNA input in every round was 200 pmol/well. The amount of coated AChE was decreased in the last four rounds in order to increase the stringency of the selection (Tab 1).

Tab 1. Protein and RNA input into each SELEX-round. Both protein (AChE) and RNA were added to each well in a total volume of 200 µL. AChE denoted the amount added to coat the well, it did not represent the total protein adsorbed.

In vitro selection of aptamer binding to AChE

After 9 cycles of selection and amplification, a new dark band (Fig1, lane d) was clearly observed. The formation of AChE-RNA complex indicated that aptamers directed to the human RBC AChE had been successfully sorted out.

Fig 1. Gel mobility shift assay for G₀ and G₉ RNA. a) G₀ RNA; b) G₀ RNA incubated with AChE 2.25 µmol/L; c) G₉ RNA; d) G₉ RNA incubated with AChE 2.25 µmol/L.

Concentration-dependence of AChE-RNA formation Various concentrations of AChE were reacted with G₉ RNA (1.2 nmol/L). It could be seen that the AChE-RNA complex bands become darker and darker (Fig 2) along with the increasing concentrations of AChE. It suggested that the AChE-RNA complex formation was concentration-dependent.

Fig 2. Gel mobility shift assay for G₉ RNA incubated with various concentrations of AChE. a) no protein; b) AChE 0.38 µmol/L; c) AChE 0.75 µmol/L; d) AChE 3 µmol/L; e) AChE 6 µmol/L.

Specificity of the aptamers Radiolabled aptamers (1.2 nmol/L) reacted well with the hRBC AChE at the concentration of 2.25 µmol/L (Fig 3, lane c), while no binding could be seen in case of rhBChE at the same concentration (Fig 3, lane b). It indicated that the aptamers selected by SELEX were highly specific to human RBC AChE.

Fig 3. Specificity of G₉ RNA aptamers to human AChE. Gel mobility shift assay was used to examine the protein-aptamer interaction. a) G₉ RNA 1.2 nmol/L; b) G₉ RNA 1.2 nmol/L+rhBChE 2.25 mmol/L; c) G₉ RNA 1.2 nmol/L+hRBC AChE 2.25 µmol/L.

Inhibition of hRBC AChE activity by RNA aptamers AChE activity was inhibited along with the increasing G₉ RNA aptamer concentrations. G₀ RNA did not show any inhibition on AChE activity (Fig 4).

Fig 4. Inhibition of rhRBC AChE activities by G₀ and G₉ RNA aptamers. n=3 independent experiments in duplicate. Mean±SD. ^bP<0.05, ^cP<0.01 vs G₉ aptamer control. Open boxes: G₀ aptamers; Black boxes: G₉ aptamers.

DISCUSSION

In this paper, we used in vitro selection and amplification to identify the RNA aptamers that bind to the human RBC AChE.

The critical step in SELEX is how to sort out the aptamer-target complex from the reaction mixture. There are several ways to separate aptamer-target complex from unbound fractions. Using a 96-well microtiter plate coated with the protein target is the simplest way in trapping of the complex. It lays out the basis for the high thoughput SELEX^[6].

Aptamer-based diagnostic reagents rivaling antibodies may be used in clinic owing to the high affinity and specifity of aptamer^[9]. Aptamers directed to AChE can be used in immunohistochemistry and Western blotting for AChE detection^[10].

The amino acid sequence of AChE in nervous system is distinct from that on the red blood cell membrane only at the C-terminus of the enzyme, the last 43 residues. The aptamers inhibiting hRBC AChE will inhibit neuronal AChE since they share an identical active center. In view of that anti-cholinesterases have been the first choice in the therapy of Alzheimer's disease and Myasthenia gravis^[11,12], the specific aptamers to AChE will be used as therapeutic or dignostic agents in the future.

ACKNOWLEDGMENT To Dr LIAO Jian (University of Bern, Switzerland) for his gift of human RBC membrane AChE.

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