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Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus

Aptamer generation using the SELEX process.(A) Seven M urea 8% denatured polyacrylamide gel electrophoresis was used to confirm ssDNA, which was separated by asymmetric PCR after each round. Lane 1: pUC18 DNA/Msp l marker. Lane 2: initial library Gp30. Lane 3: unequal length PCR products. Lane 4: target ssDNA. (B) Selected pool enrichment was monitored via indirect ELISA. (C-D) Secondary structures of aptamers A1 (C) and A4 (D) predicted by RNAstructure 5.7 software (Mathews lab, http://rna.urmc.rochester.edu/RNAstructure.html).
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pone.0140733.g001: Aptamer generation using the SELEX process.(A) Seven M urea 8% denatured polyacrylamide gel electrophoresis was used to confirm ssDNA, which was separated by asymmetric PCR after each round. Lane 1: pUC18 DNA/Msp l marker. Lane 2: initial library Gp30. Lane 3: unequal length PCR products. Lane 4: target ssDNA. (B) Selected pool enrichment was monitored via indirect ELISA. (C-D) Secondary structures of aptamers A1 (C) and A4 (D) predicted by RNAstructure 5.7 software (Mathews lab, http://rna.urmc.rochester.edu/RNAstructure.html).

Mentions: We used a microwell plate method to perform the SELEX procedure. For the subsequent round of selection, asymmetric PCR was conducted to separate the target ssDNA, which was identified using 7M urea 8% denatured polyacrylamide gel electrophoresis (Fig 1A). Selected pool enrichment resulted in the evolution of potential aptamer candidates, which were investigated using indirect ELISA. First, ssDNAs from each round were labeled with biotin; then, using streptavidin-conjugated horseradish peroxidase, the biotinylated aptamers reacted with BACE1 via ELISA. Initial library Gp30 and ssDNA obtained from the 1st, 3rd, 5th and 7th rounds were applied to perform the assay. Absorbance was increased as the selection process proceeded, which suggested enrichment in the 7th round (Fig 1B). The ssDNA obtained from the final round was amplified, cloned, and sequenced. Two sequences were identified based on their primary sequence similarity and were regarded as potential aptamer candidates (A1 and A4) (Table 2). Both sequences had a primarily stem-loop structure as indicated by the secondary structure analysis (Fig 1C and 1D).


Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

Liang H, Shi Y, Kou Z, Peng Y, Chen W, Li X, Li S, Wang Y, Wang F, Zhang X - PLoS ONE (2015)

Aptamer generation using the SELEX process.(A) Seven M urea 8% denatured polyacrylamide gel electrophoresis was used to confirm ssDNA, which was separated by asymmetric PCR after each round. Lane 1: pUC18 DNA/Msp l marker. Lane 2: initial library Gp30. Lane 3: unequal length PCR products. Lane 4: target ssDNA. (B) Selected pool enrichment was monitored via indirect ELISA. (C-D) Secondary structures of aptamers A1 (C) and A4 (D) predicted by RNAstructure 5.7 software (Mathews lab, http://rna.urmc.rochester.edu/RNAstructure.html).
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4608735&req=5

pone.0140733.g001: Aptamer generation using the SELEX process.(A) Seven M urea 8% denatured polyacrylamide gel electrophoresis was used to confirm ssDNA, which was separated by asymmetric PCR after each round. Lane 1: pUC18 DNA/Msp l marker. Lane 2: initial library Gp30. Lane 3: unequal length PCR products. Lane 4: target ssDNA. (B) Selected pool enrichment was monitored via indirect ELISA. (C-D) Secondary structures of aptamers A1 (C) and A4 (D) predicted by RNAstructure 5.7 software (Mathews lab, http://rna.urmc.rochester.edu/RNAstructure.html).
Mentions: We used a microwell plate method to perform the SELEX procedure. For the subsequent round of selection, asymmetric PCR was conducted to separate the target ssDNA, which was identified using 7M urea 8% denatured polyacrylamide gel electrophoresis (Fig 1A). Selected pool enrichment resulted in the evolution of potential aptamer candidates, which were investigated using indirect ELISA. First, ssDNAs from each round were labeled with biotin; then, using streptavidin-conjugated horseradish peroxidase, the biotinylated aptamers reacted with BACE1 via ELISA. Initial library Gp30 and ssDNA obtained from the 1st, 3rd, 5th and 7th rounds were applied to perform the assay. Absorbance was increased as the selection process proceeded, which suggested enrichment in the 7th round (Fig 1B). The ssDNA obtained from the final round was amplified, cloned, and sequenced. Two sequences were identified based on their primary sequence similarity and were regarded as potential aptamer candidates (A1 and A4) (Table 2). Both sequences had a primarily stem-loop structure as indicated by the secondary structure analysis (Fig 1C and 1D).

Bottom Line: We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model.We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment.Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

ABSTRACT
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

No MeSH data available.


Related in: MedlinePlus