A neutral pH thermal hydrolysis method for quantification of structured RNAs

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Biology

Background:

• Riboswitch aptamers exhibit complex tertiary structures with significant secondary structure content.
• Standard methods for calculating extinction coefficients underestimate RNA concentrations due to hypochromicity effects in these structures.

Purpose of the Study:

• To develop a practical and accurate method for quantifying riboswitch RNA concentrations.
• To address the systematic underestimation of RNA concentrations caused by secondary structure-induced hypochromicity.

Main Methods:

• Utilized thermal hydrolysis to generate a pool of mononucleotides from riboswitch RNAs.
• Employed precisely measured extinction coefficients for mononucleotides.
• Validated RNA concentration accuracy using quantitative 31P-NMR calibrated to an external standard.

Main Results:

• The developed method accurately quantifies riboswitch RNA concentrations.
• Thermal hydrolysis provides a reliable alternative to standard methods that suffer from underestimation.
• The procedure is effective without nucleases, fluorescent dyes, or calibration curves.

Conclusions:

• Thermal hydrolysis offers a simple and accurate method for quantifying riboswitch RNAs.
• This technique is broadly applicable for accurate quantification of various sequence-defined RNA samples.
• Precise RNA quantification is crucial for in vitro binding and kinetic assays.