Abstract
The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 104 M−1 has been assessed through a Stern–Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.
Highlights
Human serum albumin (HSA) is the most abundant globular protein in the plasma, accounting for 75% of the colloid osmotic pressure of blood [1–5]
In order to explore the structure of the Human Serum Albumin (HSA)/miR4749 complex, Förster Resonance Energy Transfer (FRET) was employed as a guide to the localization of the binding site
Atto390 was chosen because its absorbance overlaps the emission spectrum of Trp214 [50], a condition required for the dipole–dipole coupling enabling FRET
Summary
Human serum albumin (HSA) is the most abundant globular protein in the plasma, accounting for 75% of the colloid osmotic pressure of blood [1–5]. The most exploited feature of HSA resides in its versatility as a transport protein, since it is able to non-covalently bind and deliver to its targets an extraordinarily diverse range of ligands, varying from long chain fatty acids, hormones, nutrients [8] and metal ions [9], to bile acids, nitric acid and endotoxin [6], inter alia. MicroRNAs (miRNAs) are short (average of 22 nucleotides) noncoding single stranded RNAs, which can post-transcriptionally regulate the expression of genes controlling fundamental cell life processes, such as cell proliferation, differentiation, stress response and apoptosis. They induce translational repression or activation through the binding of target messenger RNAs [14–20]
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