We employed modified glass nanocapillaries to investigate interactions between the RNA-binding protein, known as cell carcinoma antigen recognized by T cells-3 (SART3), and the noncoding spliceosome component, U6 small nuclear RNA (snRNA), at the single-molecule level. We functionalized the nanocapillaries with U6 snRNA fragments, which were hybridized to DNA molecules and then covalently attached to the nanocapillary surface. When transported through the modified nanocapillaries, two different SART3-derived constructs, HAT-RRM1-RRM2 and RRM1-RRM2, exhibited resistive ionic current pulses with different dwell times, which represented their different binding affinities to tethered U6 snRNAs. The dissociation constants (KD), estimated from the bias voltage dependence of translocation events, were approximately 1.9 μM and 201 μM for HAT-RRM1-RRM2 and RRM1-RRM2, respectively. These values were comparable to corresponding values obtained with isothermal titration calorimetry, demonstrating that the modified glass nanocapillaries are applicable to analyses of protein-ligand interactions at the single-molecule level.
Bibliographical noteFunding Information:
This work was financially supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grants 2016R1A2B3011980 and 2012R1A4A1029061) to K.-H.Y., Grant NRF-2015R1A2A2A04005596 to N.-k.K. and by grants from the Global Research Laboratory Program of the Ministry of Science, ICT and Future Planning (Grant NRF-2011-0021713) to E.E.K.
© 2017 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry