DEVELOPMENT OF DNA APTAMER SEQUENCE AS BIOSENSOR DETECTING NT-PROBNP A BIOMARKER OF HEART FAILURE

• Main Author: Marcius, Donny
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/84703
• Institution: Institut Teknologi Bandung

Heart failure disease requires a diagnosis as soon as possible to prevent patient death. NT-proBNP is a stable biomarker in blood that can be used to specifically detect heart failure. Diagnostic kits for detecting NT-proBNP in the market nowadays utilise antibodies as bioreceptors. On the other hand, aptamers, singlestranded oligonucleotides, offer various advantages compared to antibodies such as lower cost of production and stability. The NT-proBNP aptamer obtained from the previous study still showed weakness in ta structure parts which do not contribute to complex formation. This research aimed to optimize the aptamer sequence to obtain a new sequence that can interact stronger and more stably with NT-proBNP without any non-essential structures. The methods comprise aptamer sequence optimization through computational modification, in silico evaluation, and in vitro experiments for confirmation. Aptamer from SELEX is optimized utilizing sequence truncation and structure similarity-guided random mutations. Binding strength is evaluated by molecular docking score as well as interaction profiles and molecular dynamics simulation is used to assess the stability of aptamer-protein complex. A colorimetric sensor based on gold nanoparticles (AuNPs) aggregation assay is used to confirm the results in vitro. The improvement of aptamer binding strength to NT-proBNP was demonstrated by the more negative values of docking score (?68.8 ± 24.1 to ?91.1 ± 5.5) and the MM/GBSA’s free binding energy (?538.5 ± 67.7 kJ/mol to ?580.1 ± 53.3 kJ/mol). A fifty nanoseconds molecular dynamics’ structural and energetic stability analysis revealed more robust and stable interactions during binding with NT-proBNP. After performing assay conditions optimization (50 ?L of 50 mM NaCl and a concentration of 0.5 ?M for the SELEX-obtained aptamer and 5 ?M for the in silico-optimized aptamer), the AuNP aggregation assay indicated that the limit of detection for detecting NTproBNP using the in silico-optimized aptamer (0.5 ng/mL) was lower compared to that obtained through SELEX (2.3 ng/mL). This research successfully improved the performance of aptamer in detecting NT-proBNP through in silico sequence optimization, suggesting potential applications for enhancing protein-targeting DNA aptamers.