Interbase-FRET binding assay for pre-microRNAs
Autor: | Marcus Wilhelmsson, Jesper R. Nilsson, Malin Lemurell, Fredrik Edfeldt, Mattias Bood, Morten Grøtli, Anders Dahlén, Anna Wiktoria Wypijewska Del Nogal |
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Rok vydání: | 2021 |
Předmět: |
0301 basic medicine
Science Medicinal chemistry 010402 general chemistry 01 natural sciences Article 03 medical and health sciences Fluorescence Resonance Energy Transfer Humans Surface plasmon resonance Multidisciplinary Oligonucleotide Chemistry Ligand binding assay RNA Isothermal titration calorimetry Surface Plasmon Resonance 0104 chemical sciences Kinetics MicroRNAs Aminoglycosides 030104 developmental biology Förster resonance energy transfer Biophysics Medicine Analytical chemistry Biogenesis Function (biology) Protein Binding |
Zdroj: | Scientific Reports, Vol 11, Iss 1, Pp 1-9 (2021) Scientific Reports |
ISSN: | 2045-2322 |
Popis: | The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies. |
Databáze: | OpenAIRE |
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