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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Chemistry

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Published: February 4th, 2013

DOI:

10.3791/50225

1Department of Chemistry, University of Virginia

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.53.

However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ≈280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

Much of cell biology occurs via molecular interactions involving DNA and RNA.4 These naturally occurring nucleic acids (NAs) interact with one another,5 with proteins,6 and with a host of small-molecule compounds and ligands in vivo (e.g., divalent cations7). The interactions may be short- or long-lived (kinetically), may range from high to moderate to low affinity (thermodynamic strength), and can also exhibit substantial variation in chemical properties and specificity - some associations are quite specific (e.g., DNA···transcription factors, RNA··&midd....

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1. Benedict's Assay for Reducing Sugars

  1. Prepare a suitable quantity of Benedict's reagent - 940 mM anhydrous sodium carbonate, 588 mM sodium citrate dehydrate, 68 mM copper (II) sulfate pentahydrate. This reagent can be stored at room temperature (RT) for at least six months with no noticeable change in reactivity.
  2. The above reagent is 6x. Thus, for 600 μl reactions, add 100 μl of Benedict's reagent to a clean 1.5 ml microcentrifuge tube (e.g., Eppendorf brand), per sample to be assay.......

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Results are shown in Figure 3 for application of these colorimetric assays to known reference compounds. Representative qualitative data are shown for the Benedict's (a), Bial's orcinol (b), and Dische's diphenylamine (c) assays, and standard curves for these three assays are shown in Figure 4. In panels 3(a-c), the left panels show positive/negative control experiments using suitably reactive/unreactive analytes; these visual results are shown in situ, in the cuvettes as descri.......

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The colorimetric assays presented here offer a simple approach to rapidly assess the chemical nature of biomolecular mixtures, such as are encountered when purifying proteins, RNAs or complexes from whole-cell lysate in preparation for further studies. As structural biology pursues more native-like assemblies, progressively greater challenges, such as sample heterogeneity, will be posed by the intricate and multi-component complexes. Supramolecular assemblies are often only marginally stable, and their successful isolati.......

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This work was funded by the University of Virginia and the Jeffress Memorial Trust (J-971). We thank L. Columbus, K. Jain, and P. Randolph for helpful discussions and critical reading of the manuscript.

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Name Company Catalog Number Comments
Reagent or equipment Supplier/company Catalog number Comments, notes
Anhydrous sodium carbonate Fisher Scientific S263  
Sodium citrate dihydrate Sigma S-4641  
Copper (II) sulfate pentahydrate VWR VW3312-2  
Orcinol monohydrate Sigma-Aldrich O1875  
Concentrated HCl VWR BDH3030  
Ferric chloride hexahydrate Sigma F-2877  
Diphenylamine Aldrich 112763  
Glacial acetic acid Fisher Scientific A28  
Sulfuric acid Sigma-Aldrich 258105  
Ethanol Koptec V1101  
Ribose Sigma R-7500 prep at 1% w/v in H2O
Ribonucleic acid from baker's yeast (S. cerevisiae) Sigma R6750 prep at 10 mg/ml in H2O; store at -20 °C
Deoxyribonucleic acid (sodium salt), from calf thymus Sigma D1501 prep at 10 mg/ml in H2O; store at 4 °C
     

Reagents, Equipment & Safety

Materials are listed in the following table in the order in which they appear in the Protocol section. Unless otherwise noted (above), all reagents can be stored at ambient room temperature and lighting. For any items not listed below (e.g., microcentrifuge tubes), the usual make / model / variety found in a standard biochemical laboratory can be used (e.g., Eppendorf brand 1.5 ml microfuge tubes). Standard plastic microfuge tubes should be used for steps involving centrifugation (e.g., to sediment particulate material near the end of each protocol). No particular material is preferable, as long as the tubes can be sealed; the typical polypropylene tubes found in biochemistry laboratories work well. In terms of safety concerns and waste disposal, standard laboratory precautions (safety glasses, fume hoods) should be exercised in pre-paring, working with, and disposing of solutions containing concentrated acetic, hydrochloric, or sulfuric acids. For organic reagents such as orcinol or diphenylamine, nitrile gloves are preferable to the common latex (natural rubber) variety.

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