Stability of a Split Streptomycin Binding Aptamer

Abstract

Here we investigated the stability of an aptamer, which is formed by two RNA-strands and binds the antibiotic streptomycin. Molecular dynamics simulations in aqueous solution confirmed the geometry and the pattern of hydrogen bond interactions that was derived from the crystalstructure (1NTB). The result of umbrella sampling simulations indicated a favored streptomycinbinding with a free energy of ‫ܩ∆‬ ୠ୧୬ୢ°= -101.7 kJ mol -1 . Experimentally, the increase in oligonucleotide stability upon binding of streptomycin was probed by single-molecule force spectroscopy. Rate dependent force spectroscopy measurements revealed a decrease in the natural off-rate (k off-COMPLEX = 0.22 ± 0.16 s -1 ) for the aptamer-streptomycin complex compared to the aptamer having an empty binding pocket (k off-APTAMER = 0.49 ± 0.11 s -1 ). This decrease in the natural off-rate corresponds to a decrease in the Gibbs free energy of ∆∆G ୱ୦ୣୣ୰ ≈ 3.4 kJ mol -1 .The simulated binding pattern and the experimental results led to the conclusion that hydrogen-bonds between both RNA strands mainly contribute to the decrease in natural-off rate and Gibbs free energy of the aptamer system studied.

Engineering Proteins at Interfaces: From Complementary Characterization to Material Surfaces with Designed Functions

“…This method has been used to probe the potential landscape upon unfolding of proteins, [72] and it can be used to probe the presence of hydrogen bonds between binding partners. [73] b) Thelateral distribution of proteins can be measured in the topography mode.M ore than two decades ago,t he membrane protein bacteriorhodopsin adsorbed to mica in buffer solution has been imaged ( Figure 5c). [70] Meanwhile the electronics of the SFM method have been improved, allowing for routine investigations of native membrane proteins such as ion-driven rotors.…”

“…This interaction can be probed by measuring the force upon retraction of the tip (Figure b). This method has been used to probe the potential landscape upon unfolding of proteins, and it can be used to probe the presence of hydrogen bonds between binding partners …”

Engineering von Proteinen an Oberflächen: Von komplementärer Charakterisierung zu Materialoberflächen mit maßgeschneiderten Funktionen

“…Dadurch wird eine Konturkarte der Oberflächentopographie aufgenommen, ohne diese zu zerstçren. [73] b) Die laterale Anordnung von Proteinen kann im Topographiemodus gemessen werden. [68] Fürw eiche Angewandte Chemie Aufsätze Proben erreicht man typischerweise eine laterale Auflçsung zwischen 1-10 nm.…”

“…[ [72] und sie kann verwendet werden, um die Anwesenheit von Wasserstoffbrücken zwischen Bindungspartnern zu detektieren. [73] b) Die laterale Anordnung von Proteinen kann im Topographiemodus gemessen werden. Vorm ehr als zwei Jahrzehnten wurde das an Glimmer adsorbierte Membranprotein Bakteriorhodopsin in Pufferlçsung abgebildet (Abbildung 5c).…”

Unconventional Split Aptamers Cleaved at Functionally Essential Sites Preserve Biorecognition Capability

“…6 In addition, the splitting results in additional oligonucleotide termini which provide additional functional groups for multiple conjugations. SPAs have demonstrated excellent capability as the sensing element in single-molecule force spectroscopy, 9 and optical 10 and electrochemical sensing platforms 11 . Several SPAs have entered clinical trials for diagnosing cancer, inflammation and macular degeneration.…”