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. 2015 Sep 22;43(19):9564–9572. doi: 10.1093/nar/gkv944

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — The experimental workflow to determine binding and stability landscapes of fluorescent RNA aptamers. (A) A DNA mutant library was created by stitch PCR. DNA sequences encoding of the single- and double-point Spinach mutants were spotted on a glass microarray and bonded to a microfluidic polydimethylsiloxane (PDMS) chip. (B) On the chip, the DNA transcription (1), a miniaturized pull-down assay (2), and a perfusion system (3) for 640 RNA mutants were automated in separate microfluidic unit cells. The chip was mounted on a thin glass coated with indium tin oxide (ITO) for control of the temperature on the glass surface. (C) Fluorescent signals of two Spinach mutants in the pull-down area of a chip unit cell. The total amount of RNA in the pull-down area was quantified with a probe molecule. (D) The binding and melting curves were used to calculate the free energy (ΔG), and enthalpy (ΔH) of the binding, the apparent melting temperature of the RNA–DFHBI complex (), and the fluorescence intensity of the Spinach complexes (S) with 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI).

Inline graphic — The experimental workflow to determine binding and stability landscapes of fluorescent RNA aptamers. (A) A DNA mutant library was created by stitch PCR. DNA sequences encoding of the single- and double-point Spinach mutants were spotted on a glass microarray and bonded to a microfluidic polydimethylsiloxane (PDMS) chip. (B) On the chip, the DNA transcription (1), a miniaturized pull-down assay (2), and a perfusion system (3) for 640 RNA mutants were automated in separate microfluidic unit cells. The chip was mounted on a thin glass coated with indium tin oxide (ITO) for control of the temperature on the glass surface. (C) Fluorescent signals of two Spinach mutants in the pull-down area of a chip unit cell. The total amount of RNA in the pull-down area was quantified with a probe molecule. (D) The binding and melting curves were used to calculate the free energy (ΔG), and enthalpy (ΔH) of the binding, the apparent melting temperature of the RNA–DFHBI complex (), and the fluorescence intensity of the Spinach complexes (S) with 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI).