Accurate nucleic acid concentrations by nuclear magnetic resonance

Abstract

Determination of the concentration of biochemical samples often yields values with uncertainties of 10-20% or more. This paper details a protocol for use with 500- to 600-MHz NMR spectrometers to measure approximately 1mM concentrations within +/-1-3% accuracy. With suitable precautions, all compounds have equal NMR "absorption coefficients" for protons. About 2mg of sample are needed for proteins and nucleic acids with MW=5000, although less accurate determinations could be made with smaller amounts. The technique utilizes standardized internal reference reagent compounds, cacodylic acid or 3-(trimethylsilyl)propionic-2,2,3,3-d(4) acid sodium salt. Spectra were signal-averaged using long interpulse delays so that integrals of nonexchangeable protons could be quantified relative to the reference standard. Accurate determinations require careful optimization of the homogeneity of the magnetic field and meticulous attention to sample preparation and spectral processing. The main source of error is usually the accuracy of micropipets. If sample preparation errors could be eliminated, the lower limit of accuracy with the current generation of NMR spectrometers is probably near 0.4%. However, this would require >99.5% sample purity. Methods are described to establish the concentration of the standards, and applications are illustrated with DNA mono- and oligonucleotides. Similar procedures should apply to proteins, polysaccharides, and other biomolecules, with about the same accuracy and precision.