Mechanical tuning of molecular machines for nucleotide recognition at the air-water interface
© Mori et al; licensee Springer. 2011
Received: 12 October 2010
Accepted: 7 April 2011
Published: 7 April 2011
Molecular machines embedded in a Langmuir monolayer at the air-water interface can be operated by application of lateral pressure. As part of the challenge associated with versatile sensing of biologically important substances, we here demonstrate discrimination of nucleotides by applying a cholesterol-armed-triazacyclononane host molecule. This molecular machine can discriminate ribonucleotides based on a twofold to tenfold difference in binding constants under optimized conditions including accompanying ions in the subphase and lateral surface pressures of its Langmuir monolayer. The concept of mechanical tuning of the host structure for optimization of molecular recognition should become a novel methodology in bio-related nanotechnology as an alternative to traditional strategies based on increasingly complex and inconvenient molecular design strategies.
Water used for the subphase was distilled using an Autostill WG220 (Yamato) and deionized using a Milli-Q Lab (Millipore). Its specific resistance was greater than 18 MΩ · cm. Spectroscopic grade chloroform (Wako Pure Chemical Co., Osaka, Japan) was used as the spreading solvent. Ribonucleotides [adenosine 5'-monophosphate disodium salt (AMP), cytidine 5'-monophosphate disodium salt (CMP), guanosine 5'-monophosphate disodium salt (GMP), and uridine 5'-monophosphate disodium salt (UMP)] and lithium chloride were purchased from Wako Pure Chemical Co. (Osaka, Japan). The synthesis of the molecular machine, cholesterol-armed-triazacyclononane (1), was described previously . Isotherms of surface pressure and molecular area (π-A isotherm) were measured at 20.0°C using an FSD-300 computer-controlled film balance (USI System, Fukuoka, Japan). A period of 15 min was allowed for spreading solvent evaporation, compression was commenced at a rate of 0.2 mm s-1. Fluctuation of the subphase temperature was within ± 0.2°C.
Results and discussion
As shown in Figure 4A, the binding constants of the nucleotides to the monolayer of 1 gradually decreased as the surface pressure increased. This is because expansion of the molecular area of 1 by binding to the nucleotides is thermodynamically unfavourable at higher pressures. As will be described later, when the triazacyclononane moiety is not complexed with a central Li+ ion electrostatic interaction between 1 and the phosphate group within the nucleotide becomes less important. Hence, on the surface of pure water, there exists a rather ambiguous interaction between 1 and the base portion of the nucleotides, and this interaction is quite sensitive to other factors. Although the absolute value of binding constants decreased drastically, differences in the binding efficiencies amongst the nucleotides became obvious at higher surface pressures. For example, ratios of binding constants, K(AMP/UMP), K(CMP/UMP), and K(GMP/UMP), are 0.78, 1.05, and 0.68, respectively, at a surface pressure of 5 mN m-1, whereas K(AMP/UMP), K(CMP/UMP), and K(GMP/UMP) values become 9.89, 8.77, and 5.52, respectively, when compressed to 35 mN m-1. Thus, discrimination of GMP and UMP from AMP and CMP is possible as well as between GMP and UMP, although differentiation between AMP and CMP is rather difficult even at greater surface pressures.
Complexation of Li+ ion by the triazacyclononane ring causes two variations in the characteristics of the recognition system. The presence of Li+ ion at the core of 1 ensures strong electrostatic interaction between the monolayer and the nucleotides. In addition, the complexation of Li+ ion stabilizes the conformation of the cyclononane ring of 1, resulting in a rather simple situation of discrimination amongst the nucleotides (Figure 4B). Although the binding constants of UMP to the monolayer exhibit a distinct dependence on surface pressure, an order of binding constant (K(CMP) > K(GMP) > K (AMP)) is maintained over the entire pressure range. An apparent advantage in the Li+-containing system is due to a significant increase in the binding constant of CMP. As seen in Figure 3B, binding of CMP to the monolayer of 1 does not require large expansion of the monolayer in contrast to AMP and GMP (Figure 3A, C). This binding mode should provide more favourable binding to the molecular assembly. On the other hand, the binding curve for UMP is unusual when compared with the other nucleotides. As has been suggested in previous research , the uridine moiety of UMP probably interacts with the cyclononane ring, thus competing with the major interactions between the phosphate and the Li+ ion.
These results clearly indicate that the recognition of aqueous nucleotides can be tuned both by the surface pressure and the presence of Li+ ion, although the same recognition element (1) was used throughout this investigation. The optimum discrimination between nucleotides can be obtained as follows, where the maximum ratio of binding constants and the conditions applied are summarized: K(CMP/AMP) = 6.5 ([Li+] = 10 mM and π = 5 mN · m-1); K(CMP/GMP) = 3.11 ([Li+] = 10 mM and π = 20 mN · m-1); K(CMP/UMP) = 8.77 ([Li+] = 0 mM and π = 35 mN · m-1); K(AMP/GMP) = 2.22 ([Li+] = 0 mM and π = 20 mN · m-1); K(AMP/UMP) = 9.89 ([Li+] = 0 mM and π = 35 mN · m-1); K(GMP/UMP) = 5.52 ([Li+] = 0 mM and π = 35 mN · m-1). On the other hand, the maximum binding constants for individual nucleotides are: K(CMP) = 1080 M-1 ([Li+] = 10 mM and π = 5 mN · m-1); K(AMP) = 550 M-1 ([Li+] = 0 mM and π = 5 mN · m-1); K(GMP) = 480 M-1 ([Li+] = 0 mM and π = 5 mN · m-1); K(UMP) = 710 M-1 ([Li+] = 0 mM and π = 5 mN · m-1). Therefore, conditions suitable for discrimination of the nucleotides and for most efficient binding of a single nucleotide component can be selected. The molecular recognition system presented here is therefore distinct different from more conventional ones where the structure of recognition components primarily defines binding efficiency of guest molecules.
Prior to this and our other preliminary reports, discrimination of nucleotides has not been easy to achieve because of their structural similarity, and despite its importance in biological and pharmaceutical fields. This research strikingly demonstrates a method for molecular discrimination amongst structurally similar nucleotides by mechanical tuning of a simple host at a dynamic interfacial medium. Recognition and discrimination of ribonucleotides can also be optimized. The concept of mechanical tuning for optimization of molecular recognition should become a novel methodology in bio-related nanotechnology as an alternative to traditional strategies based on increasingly complex and inconvenient molecular design strategies.
adenosine 5'-monophosphate disodium salt
cytidine 5'-monophosphate disodium salt
guanosine 5'-monophosphate disodium salt
uridine 5'-monophosphate disodium salt.
This work was partly supported by World Premier International Research Center Initiative (WPI Initiative), MEXT, Japan and Core Research for Evolutional Science and Technology (CREST) program of Japan Science and Technology Agency (JST), Japan.
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