Semmelweis University, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry
Introduction: Aptamers are oligonucleotides that can bind to their targets with similar selectivity and affinity to those of antibodies. Nucleic acid aptamers are promising tools of diagnostics and therapeutics for their numerous advantages and wide range of utility.
Brain natriuretic peptide (BNP) is expressed and stored in myocardial tissue and released into circulation upon increased tension playing role in blood pressure regulation. An elevated serum level of BNP and inactive amino terminal fragment of BNP pro-hormone (NT-proBNP) could be measured in heart failure patients and they are useful biomarkers for diagnosis of left-ventricular systolic dysfunction. Current serum BNP level measurement methods are based on various immunoassay technics.
Aims: Aptamers for the N terminal end of the NT-proBNP protein were selected previously in our laboratory. We aimed to select aptamers for another epitope of the NT-proBNP protein to aid the development of proBNP specific sandwich assays.
Method: To obtain the appropriate aptamers, peptides molecules from the C terminal end of proBNP and in vitro translated proBNP protein were alternately used as target molecules during the seven selection steps of SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Elimination of nonspecific aptamers was implemented with three different negative selection steps. Last step of selection was followed by sequencing of 96 aptamer candidates. Screening of individual aptamer candidates were accomplished by using AlphaScreen based assay. Functional analysis of the most auspicious aptamers was evaluated in human plasma samples to monitor their diagnostic utility.
Results: The analyzed aptamer candidates possessed significantly higher affinity to their target molecules in comparison to control aptamers. Furthermore, some of the isolated aptamers remained to be functional even in human plasma demonstrating their high selectivity.
Conclusion: Selected aptamers showed promising results under condition of the intended application. The proBNP specific aptamer pairs enable us to establish oligonucleotide sandwich based assay for proBNP detection.
Supported BY the ÚNKP-17-3-III-SE-25 New National Excellence Program of the Ministry of Human Capacities.
Doctoral School: Molecular Medicine
Supervisor: Tamás Mészáros
E-mail address: firstname.lastname@example.org