Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries

Choongman Lee; Joon Kyu Park; Yeoan Youn; Joo Hyoung Kim; Kyo Seok Lee; Nak Kyoon Kim; Eunji Kim; Eunice Eunkyeong Kim; Kyung Hwa Yoo

doi:10.1021/acs.analchem.6b04245

Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries

Choongman Lee, Joon Kyu Park, Yeoan Youn, Joo Hyoung Kim, Kyo Seok Lee, Nak Kyoon Kim, Eunji Kim, Eunice Eunkyeong Kim, Kyung Hwa Yoo

Department of Physics

Research output: Contribution to journal › Article › peer-review

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Abstract

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 (K_D), 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.

Original language	English
Pages (from-to)	2390-2397
Number of pages	8
Journal	Analytical Chemistry
Volume	89
Issue number	4
DOIs	https://doi.org/10.1021/acs.analchem.6b04245
Publication status	Published - 2017 Feb 21

Bibliographical note

Funding 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.

Publisher Copyright:
© 2017 American Chemical Society.

All Science Journal Classification (ASJC) codes

Analytical Chemistry

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10.1021/acs.analchem.6b04245

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title = "Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries",

abstract = "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.",

author = "Choongman Lee and Park, {Joon Kyu} and Yeoan Youn and Kim, {Joo Hyoung} and Lee, {Kyo Seok} and Kim, {Nak Kyoon} and Eunji Kim and Kim, {Eunice Eunkyeong} and Yoo, {Kyung Hwa}",

note = "Funding 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. Publisher Copyright: {\textcopyright} 2017 American Chemical Society.",

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AU - Lee, Choongman

AU - Park, Joon Kyu

AU - Youn, Yeoan

AU - Kim, Joo Hyoung

AU - Lee, Kyo Seok

AU - Kim, Nak Kyoon

AU - Kim, Eunji

AU - Kim, Eunice Eunkyeong

AU - Yoo, Kyung Hwa

N1 - Funding 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. Publisher Copyright: © 2017 American Chemical Society.

PY - 2017/2/21

Y1 - 2017/2/21

N2 - 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.

AB - 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.

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Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries

Abstract

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