Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1

Nucleic acid aptamers are a class of molecular recognition elements capable of target interaction at high specificity and affinity. This study was aimed to generate an RNA aptamer against the Human Pituitary Tumour Transforming Gene 1 (PTTG1) oncoprotein which can serve as a potential diagnostic and...

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Main Author:	Sankar, Prabu Siva
Format:	Thesis
Language:	English
Published:	2019
Subjects:	R5-920 Medicine (General)
Online Access:	http://eprints.usm.my/46406/1/PRABU%20SIVA%20SANKAR_HJ.pdf
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spelling	my-usm-ep.464062020-03-02T02:31:17Z Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1 2019-07 Sankar, Prabu Siva R5-920 Medicine (General) Nucleic acid aptamers are a class of molecular recognition elements capable of target interaction at high specificity and affinity. This study was aimed to generate an RNA aptamer against the Human Pituitary Tumour Transforming Gene 1 (PTTG1) oncoprotein which can serve as a potential diagnostic and therapeutic tool. PTTG1 gene was first cloned into the pET14b vector to incorporate the small 6X Histidine tag. Protein expression and purification ensued with the E. coli Rosetta 2(DE3)pLysS strain under the urea denaturing method. The protein identity was confirmed by western blot using monoclonal anti-PTTG1 antibody. Intrinsic florescence spectra analysis and GST-pulldown interaction analysis with GST-p53 conferred signs of successful protein refolding. 11 SELEX cycles with dual partitioning strategy were performed with positive indication of enrichment. Sequencing of the cycle 8 pool revealed a putative aptamer (SECURA-3) which depicted binding to PTTG1. SECURA-3 had a dissociation constant estimated at 16.41 ± 6.4 nM. SECURA-3 was employed for development of aptamer-based diagnostic assays namely ALISA, aptamer-based dot blot, aptamer based western blot and aptahistostaining. SECURA-3 was extended with a poly A-tail at the 3’-end to functionally labelled with the biotin by hybridization with the biotin labelled oligo dT sequence. SECURA-3 aptamer demonstrated detection of up to 0.0256 nM PTTG1 in the direct ALISA assay and with the sandwich configuration, SECURA-3 demonstrated detection of PTTG1 up to 3.2 nM with the aptamer as the capturing agent and 16 nM with the aptamer as the detection agent. 2019-07 Thesis http://eprints.usm.my/46406/ http://eprints.usm.my/46406/1/PRABU%20SIVA%20SANKAR_HJ.pdf application/pdf en public phd doctoral Universiti Sains Malaysia Institut Perubatan & Pergigian Termaju
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	R5-920 Medicine (General)
spellingShingle	R5-920 Medicine (General) Sankar, Prabu Siva Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1
description	Nucleic acid aptamers are a class of molecular recognition elements capable of target interaction at high specificity and affinity. This study was aimed to generate an RNA aptamer against the Human Pituitary Tumour Transforming Gene 1 (PTTG1) oncoprotein which can serve as a potential diagnostic and therapeutic tool. PTTG1 gene was first cloned into the pET14b vector to incorporate the small 6X Histidine tag. Protein expression and purification ensued with the E. coli Rosetta 2(DE3)pLysS strain under the urea denaturing method. The protein identity was confirmed by western blot using monoclonal anti-PTTG1 antibody. Intrinsic florescence spectra analysis and GST-pulldown interaction analysis with GST-p53 conferred signs of successful protein refolding. 11 SELEX cycles with dual partitioning strategy were performed with positive indication of enrichment. Sequencing of the cycle 8 pool revealed a putative aptamer (SECURA-3) which depicted binding to PTTG1. SECURA-3 had a dissociation constant estimated at 16.41 ± 6.4 nM. SECURA-3 was employed for development of aptamer-based diagnostic assays namely ALISA, aptamer-based dot blot, aptamer based western blot and aptahistostaining. SECURA-3 was extended with a poly A-tail at the 3’-end to functionally labelled with the biotin by hybridization with the biotin labelled oligo dT sequence. SECURA-3 aptamer demonstrated detection of up to 0.0256 nM PTTG1 in the direct ALISA assay and with the sandwich configuration, SECURA-3 demonstrated detection of PTTG1 up to 3.2 nM with the aptamer as the capturing agent and 16 nM with the aptamer as the detection agent.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Sankar, Prabu Siva
author_facet	Sankar, Prabu Siva
author_sort	Sankar, Prabu Siva
title	Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1
title_short	Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1
title_full	Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1
title_fullStr	Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1
title_full_unstemmed	Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1
title_sort	elucidating the diagnostic and therapeutic potential of an rna aptamer against human pituitary tumour transforming gene 1
granting_institution	Universiti Sains Malaysia
granting_department	Institut Perubatan & Pergigian Termaju
publishDate	2019
url	http://eprints.usm.my/46406/1/PRABU%20SIVA%20SANKAR_HJ.pdf
_version_	1747821663416745984

Elucidating The Diagnostic And Therapeutic Potential Of An RNA Aptamer Against Human Pituitary Tumour Transforming Gene 1

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