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

Summary

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

Abstract

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).

Introduction

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···splicing factors), while other interactions are necessarily far more generic (e.g., DNA···bacterial histone-like HU proteins⁸). Non-specific interactions with NAs can have practical consequences for in vitro experiments involving mixtures of biomolecules, as it is possible, and even likely, that some NAs will associate with the biomolecules of interest, at least under some subset of the experimental conditions being used (ionic strength, solution pH, etc).

Consider, for instance, production of a protein of interest (POI) via heterologous over-expression of the recombinant protein in Escherichia coli cell culture; such a procedure is routinely performed in virtually any structural biology lab.⁹ In preparing for further experiments, such as biochemical/biophysical characterization, crystallization, etc., initial efforts generally focus on obtaining a sufficient quantity of the POI in as pure a form as possible, ideally as a chemically homogeneous and biophysically monodisperse specimen. After disruption of host cells, the early stages of a typical purification workflow aim to isolate the POI from E. coli proteins, nucleic acids, cell wall debris, and other components of the cellular lysate. However, host NAs may co-purify with the POI for several physicochemical reasons - a highly basic POI may non-specifically pull-down host DNA/RNA; the POI may have a generic NA-binding activity (e.g., the aforementioned HU); the POI may be a fairly specific NA-binding protein but exhibit cross-reactivity with host RNAs or DNAs; host NAs may interact with a chromatography matrix and thereby simply co-elute with the POI; and so on. Indiscriminate, high-affinity binding of host NAs to a POI can pose a vexing problem because the NA impurities will likely interfere with downstream experiments (e.g., fluorescence anisotropy assays of POI•RNA binding¹⁰). Alternatively, unanticipated POI···NA associations also can be viewed fortuitously, as such interactions illuminate the POI's nucleic acid-binding capacity. Either way, whether NAs are key components or contaminants, one must first quantify and identify the type (DNA, RNA) of co-purifying NAs in preparation for downstream experiments.

Several analytical methods exist for detecting and quantitating NAs in a sample. Most of the available methods are fundamentally either spectrophotometric (e.g., A₂₆₀ absorbance values and A₂₆₀/A₂₈₀ ratios), fluorometric (e.g., binding of thiazole orange or other fluorescent dyes to NA), or colorimetric (susceptibility of nucleosides to chemical reactions that yield chromophores absorbing in the uv-vis region of the electromagnetic spectrum), as recently described by De Mey et al.¹ However, the crucial step of identifying the type of polynucleotide as RNA or DNA is beyond the scope of many of these quantitation approaches. Here we provide a set of colorimetric assays to rapidly identify the types of NA components in a proteinaceous sample.

The protocols described here can be efficiently executed without additional steps of isolating the potential NA impurities, and rely on Benedict's assay for reducing sugars¹¹, the orcinol assay for pentoses^12,13, and diphenylamine reactions^14,15 of 2'-deoxypentoses (Figures 1 and 2). The Benedict's test (Figure 2a) utilizes the ability of the linear, open-chain (aldehyde) form of an aldose sugar to reduce Cu²⁺, with concomitant oxidation of the sugar's carbonyl to a carboxylate moiety and production of Cu₂O as an insoluble red precipitate. This reaction will test positive with free reducing sugars such as aldoses and ketoses (which convert to the corresponding aldoses via enediol intermediates), but not with pentose sugars that are locked into cyclic form as part of the covalent backbone of a DNA or RNA polynucleotide. Because of the minimalistic requirement of a free hemiacetal functionality, other compounds that could test positive in this assay - and therefore act as potential interferents - include α-hydroxy-ketones and short oligosaccharides (e.g. the disaccharide maltose). Both the Bial's orcinol (Figure 2b) and Dische's diphenylamine (Figure 2c) reactions are based on initial destruction of the polynucleotide backbone, via depurination of the nucleoside and further acid- or base-catalyzed hydrolysis of the parent nucleotides, to yield furan-2-carbaldehyde (furfural) derivatives; these derivatives then react with either a polyhydroxy phenol such as orcinol (Bial's) or diphenylamine (Dische's) reagents to form colored condensation products of largely unknown chemical structure. The DNA versus RNA specificity of the Dische's assay stems from the fact that the pentose sugar must be 2'-deoxygenated in order to be susceptible to oxidation to ω-hydroxylevulinyl aldehyde, which further reacts with diphenylamine under acidic conditions to yield a bright blue condensate (Figure 2c). Using the streamlined protocols described here, we have found that these sugar-specific colorimetric reactions can differentiate between RNA and DNA, and will also indicate the presence of free reducing sugars such as glucose, fructose, or ribose in a biomolecular sample.

Protocol

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 assayed.
3. Add anywhere from 10 μl to 500 μl of sample to this tube; the optimal volume can be determined based on the intensity of color formation in an initial trial run. If sufficient sample is available then begin such trials at the maximal possible sample volume (i.e., five-sixths of the overall reaction volume, 500 μl of sample in this case), and then dilute in subsequent assays.
4. Add ddH₂O to the tube to bring the final volume to 600 μl; mix the solution by vortexing or pipetting.
5. Incubate the samples for 20 min in a boiling water bath.
6. Remove the heated sample from the bath and allow it to cool at RT for 10 min.
7. Centrifuge the sample tube at > 9,300 x g (~10,000 rpm in an FA45-24-11 Eppendorf fixed-angle rotor) for 5 min in order to sediment any particulate material; this step is more important for quantitative rather than qualitative studies.
8. Aliquot the supernatant from this tube into a clean cuvette.
9. Blank the uv-vis spectrophotometer with water.
10. Measure the absorbance of this sample at 475 nm.

2. Bial's Orcinol Assay for Pentose Sugars

1. Prepare a suitable quantity of fresh Bial's reagent - 24.2 mM orcinol monohydrate (see Figure 2b for the structure of this compound), 6 M HCl, 0.025% w/v ferric chloride hexahydrate. Note: For extended storage, the Bial's reagent can be prepared as two separate stock solutions: (i) Reagent A [0.05% w/v FeCl3•6H₂O in concentrated HCl] and (ii) Reagent B [422 mM orcinol monohydrate prepared in 95% ethanol]. Reagent A can be stored at RT for six months; Reagent B can be stored at 4 °C for one month, covered with foil to limit light exposure. These stock solutions are mixed in a 15 (A) : 1 (B) v/v ratio prior to use.
2. The above reagent is 2x. Thus, for 1.0 ml reactions, add 500 μl of Bial's reagent to a clean 1.5 ml microcentrifuge tube, per sample to be assayed.
3. Add anywhere from 10 μl to 500 μl of sample to this tube. As per note 1.3 (above), if sufficient sample is available then begin trial reactions using the maximal possible sample volume (i.e., one-half the overall reaction volume, 500 μl of sample in this case) and dilute from there.
4. Add ddH₂O to the tube to bring the final volume to 1.0 ml; mix the solution by vortexing or pipetting.
5. Incubate the samples for 20 min in a boiling water bath.
6. Remove the heated sample from the bath and allow it to cool at RT for 10 min.
7. Centrifuge the sample tube at > 9,300 x g (~10,000 rpm in an FA45-24-11 Eppendorf fixed-angle rotor) for 5 min in order to sediment any particulate material; this step is more important for quantitative rather than qualitative studies.
8. Aliquot the supernatant from this tube into a clean cuvette for visual inspection.
9. For semi-quantitative analysis, blank the uv-vis spectrophotometer with water and measure the absorbance of the cuvette sample at 660 nm.

3. Dische's Diphenylamine Assay for 2'-deoxypentose Sugars

1. Prepare a suitable quantity of Dische's diphenylamine reagent - 60 mM diphenylamine, 11 M glacial acetic acid, 179 mM sulfuric acid, 0.62% v/v ethanol. This reagent can be prepared in advance and stored at RT in a dark container, or covered with foil, to limit light exposure. Due to light sensitivity the reagent should not be stored indefinitely, though in practice it can be prepared every two to three months with no apparent change in reactivity.
2. The above reagent is 2x. Thus, for 1.0 ml reactions, add 500 μl of Dische's reagent to a clean 1.5 ml microcentrifuge tube, per sample to be assayed.
3. Add anywhere from 10 μl to 500 μl of sample to this tube. As per note 1.3 (above), if sufficient sample is available then begin trial reactions using the maximal possible sample volume (i.e., one-half the overall reaction volume, 500 μl of sample in this case) and dilute from there.
4. Add ddH₂O to the tube to bring the final volume to 1.0 ml; mix the solution by vortexing or pipetting.
5. Incubate the samples for 20 min in a boiling water bath.
6. Remove the heated sample from the bath and allow it to cool at RT for 10 min.
7. Centrifuge the sample tube at >9,300 x g (~10,000 rpm in an FA45-24-11 Eppendorf fixed-angle rotor) for 5 min in order to sediment any particulate material; this step is more important for quantitative rather than qualitative studies.
8. Aliquot the supernatant from this tube into a clean cuvette for visual inspection.
9. For semi-quantitative analysis, blank the uv-vis spectrophotometer with water and measure the absorbance of the cuvette sample at 600 nm.

4. Further Usage Notes

1. The following classes of molecules are suitable reference compounds for positive and negative control reactions for each assay:
   • Benedict's - Positive = free ribose, fructose, glucose; Negative = RNA, DNA, ATP, etc. (any saccharide lacking a free reducing sugar functionality)
   • Bial's - Positive = RNA (e.g. baker's yeast extract), ribose, ATP, UMP; Negative = bovine serum albumin (BSA) or any other protein
   • Dische's - Positive = DNA (e.g. calf thymus); Negative = RNA, ATP, etc. (any non-2'-deoxygenated nucleotide)
2. These assays have been found to be fairly resilient to compounds often used in protein purification; for instance, common salts such as NaCl, (NH₄)₂SO₄, and K₂SO₄ did not interfere, and the reactions appear generally unaffected by the contents of the diluent. Detergents or chaotropic agents such as urea may affect the reactivity of the assays if the pH is highly basic; a neutral to acidic pH range is generally most optimal for the Bial's and Dische's assays because of the underlying reaction chemistry (see the text and the protons in Figure 2b, c). Potentially suboptimal reaction conditions, suspected interferents, etc. should be tested on a case-by-case basis, using positive and negative control experiments with reference compounds.

Results

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...

Discussion

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...

Materials

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.