Abstract
Aptamers are functional nucleic acids that bind to a range of targets (small molecules, proteins or cells) with a high affinity and specificity. Chemically-modified aptamers are of interest because the incorporation of novel nucleobase components can enhance aptamer binding to target proteins, while fluorescent base analogues permit the design of functional aptasensors that signal target binding. However, since optimally modified nucleoside designs have yet to be identified, information about how to fine tune aptamer stability and target binding affinity is required. The present work uses molecular dynamics (MD) simulations to investigate modifications to the prototypical thrombin-binding aptamer (TBA), which is a 15-mer DNA sequence that folds into a G-quadruplex structure connected by two TT loops and one TGT loop. Specifically, we modeled a previously synthesized thymine (T) analog, namely 5-furyl-2′-deoxyuridine (5FurU), into each of the six aptamer locations occupied by a thymine base in the TT or TGT loops of unbound and thrombin bound TBA. This modification and aptamer combination were chosen as a proof-of-principle because previous experimental studies have shown that TBA displays emissive sensitivity to target binding based on the local environment polarity at different 5FurU modification sites. Our simulations reveal that the chemically-modified base imparts noticeable structural changes to the aptamer without affecting the global conformation. Depending on the modification site, 5FurU performance is altered due to changes in the local environment, including the modification site structural dynamics, degree of solvent exposure, stacking with neighboring bases, and interactions with thrombin. Most importantly, these changes directly correlate with the experimentally-observed differences in the stability, binding affinity and emissive response of the modified aptamers. Therefore, the computational protocols implemented in the present work can be used in subsequent studies in a predictive way to aid the fine tuning of aptamer target recognition for use as biosensors (aptasensors) and/or therapeutics.
Highlights
Aptamers are single-stranded DNA or RNA oligonucleotides that have the ability to recognize and bind targets with a high specificity and affinity [1,2]
To provide a basis for understanding the impact of thymine modifications on the structure and dynamics of an isolated thrombin-binding aptamer (TBA), 0.5 μs molecular dynamics (MD) simulations were initially performed on the native aptamer starting from the structure of the unbound aptamer extracted from a crystal structure corresponding to the human α-thrombin–aptamer complex (Protein Data Bank (PDB) ID: 4DII) [11]
The present work highlights the impact of the fluorescent 5-furyl-2 -deoxyuridine (5FurU) base within the three unique loops of TBA on the unbound aptamer and TBA–thrombin complex. 5FurU is an excellent T-base analog, as the probe maintains the overall GQ structure of TBA in the presence and absence of the target protein
Summary
Aptamers are single-stranded DNA or RNA oligonucleotides that have the ability to recognize and bind targets with a high specificity and affinity [1,2]. Noncovalent intermolecular interactions allow aptamers to adopt a range of tertiary structures with diverse functions. Aptamers have been shown to fold into unique 3D-structures, possessing a combination of loops, stems, hairpins, pseudoknots, bulges and/or G-quadruplexes. Aptamers have been designed to bind to a Molecules 2019, 24, 2908; doi:10.3390/molecules24162908 www.mdpi.com/journal/molecules Molecules FOR PEER REVIEW 24, x2908. 22of of 18 hairpins, pseudoknots, bulges and/or G-quadruplexes. Aptamers have been designed to + and Cu2+. ), dyes green), ligands bind a broad spectrum of target molecules metals (ATP, AMP, and arginamide), antibiotics (tetracycline, tobramycin, and neomycin), vitamins (B12 ), ligands (ATP, AMP, and arginamide), antibiotics (tetracycline, tobramycin, and neomycin), vitamins proteins (thrombin(thrombin and cytochrome hormones (progesterone),
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