Hydrolysis of Nucleic Acids

EXPERIMENT NO. 9

HYDROLYSIS OF NUCLEIC ACIDS

Abstract

This experiment intended to study the acid and base hydrolysis of DNA and RNA. Furthermore, itaimed to characterize and differentiate the hydrolysates using various qualitative tests. Hydrolysis studieswere done by the addition of HCl and NaOH to DNA and RNA samples. The systems were heated for 1hour in a boiling water bath Control samples, consisting of unhydrolyzed DNA and RNA, were alsoprepared. After the hydrolysis procedure, Bial's Test and test for purines were performed to characterizethe hydrolysates. Despite a few erroneous results, in general the experiment was successfully inproviding knowledge regarding nucleic acid hydrolysis. The qualitative tests employed were alsosuccessful in differentiating the hydrolysates.

Discussion of Data and Results

Nucleic acids are the most fundamental constituent of a living cell. They generally serve as the storehouses and carriers of genetic information. There are two types of nucleic acid: ribonucleic acid(RNA) and deoxyribonucleic acid (DNA), structures of which are as shown in figure 1.

Figure 1.

Chemical structure of RNA and DNA.

(http://scienceblogs.com/transcript/2006/11/autism_rna.php)

As shown from the figure, monomer units of each nucleotide contain a five carbon sugar: ribose in RNA, and 2'- deoxyribose in DNA. The sugar units differ only in the 2' hydroxyl group on the ribose. In the nucleic acids, two monomer units is connected through a phosphate residue attached to the hydroxyl of the 5' carbon of one unit and the 3' hydroxyl of the next one. This then forms a phosphodiester bridge between two sugar residues.

The backbone of nucleic acid is made up primarily of the phosphodiester-linked sugar residues. Each monomer carries a basic group, labeled as B in Figure 1, attached to the 1' carbon of the sugar. The nucleic acid bases are of two kinds: the purines, adenine and guanine, and the pyrimidines, cytosine,thymine (for DNA), and uracil (for RNA). The structures of these bases are given in Figure 2.

The backbone of nucleic acid is made up primarily of the phosphodiester-linked sugar residues. Each monomer carries a basic group, labeled as B in Figure 1, attached to the 1' carbon of the sugar. The nucleic acid bases are of two kinds: the purines, adenine and guanine, and the pyrimidines, cytosine, thymine (for DNA), and uracil (for RNA). The structures of these bases are given in Figure 2.

Figure 2 .

Nucleotide bases.(http://www.wvup.edu/ecrisp/fournucleotides.jpg)

DNA and RNA are polynucleotides. In principle, a polynucleotide could be generated from its monomers by elimination of water between each pair of monomers. This reaction is a condensation reaction. The reaction can be imagined as adding another nucleotide to the polynucleotide chain by a dehydration reaction. However, the reported free energy change is about +25 kJ/mol, a quite positive value. The equilibrium will then lie to the side where hydrolysis of the phosphodiester bond is favored.

Though polynucleotides are thermodynamically unstable, their hydrolysis still proceeds at a veryslow rate, unless catalyzed. Hydrolysis can either proceed by acid/base catalysis or through the action of enzymes. The site at which hydrolysis occurs could also differ. The sites of cleavage could be any of thefollowing: cleavage in the sugar, in the phosphate backbone, or in the base. The reaction then mayproduce the following: purine and pyrimidine bases, oligonucleotides, nucleosides, ribose or deoxyriboseand phosphates. In this experiment, the acid and base-catalyzed hydrolysis of DNA and RNA werestudied. The products of the hydrolysis reaction (or hydrolysates) were also characterized anddifferentiated using Bial's test and test for purine bases.From Figure 1, it can be noticed that each phosphodiester bridge carry a net negative charge.This net negative charge makes the nucleic acid less susceptible to nucleophilic species. Nonetheless, atalkaline conditions, base-catalyzed hydrolysis may occur and proceed as follows:

Figure 3.

Base-catalyzed hydrolysis of RNA. [3]

In the base catalyzed hydrolysis of a nucleic acid, the hydroxyl ion assists the attack of the 2'hydroxyl group on the phosphorus leading to the formation of the cyclic 2',3' monophosphateintermediate. The intermediate is highly unstable. Introduction of water to form either 2' or 3' nucleosidesas products stabilizes the said species. Only RNA undergoes base hydrolysis. The absence of the 2'hydroxyl group in DNA makes it unable to form the cyclic monophosphate intermediate. DNA is thenimmune to base hydrolysis. This property yet again contributes to the stability of DNA as a molecule.Chain cleavage of RNA can also be acid catalyzed. The products would still be a racemic mixtureof 2' and 3' nucleosides. The reaction would proceed as follows:

Figure 4.

Acid-catalyzed chain cleavage of RNA. [4]

From the figure, it can been that an acid-catalyzed chain cleavage also arrives at the cyclic 2',3'-monophosphate intermediate. This would then eventually rearrange to give the 2' and 3' nucleosides asproducts.

For DNA (also RNA) acid hydrolysis cleaves the predisposed purine N-glycosyl bonds. If thenucleic acids are placed in a dilute acid solution, coupled with heating, the adenine and guanine residuesare liberated. What remains are apurinic sites. Mild heating doesn't release pyrimidines. Further heatingin sealed test tube or autoclave is required to cleave pyrimide N-glycosyl bonds. The acid-catalyzeddepurination of RNA/DNA proceeds as follows:

Figure 5 .

Acid-catalyzed depurination of nucleic acids. [4]

In the reaction mechanism given in Figure 5, it can be deduced that the depurinationoccurs is promoted by the protonation of the purine base, thus, weakening the N-glycosyl bond. The N-glycosyl bond is then irreversibly broken by the neighboring oxygen atom giving a free purine base and anapurinic nucleic acid. Acid-catalyzed depyrimidization also proceed in a similar mechanism asdepurination. Depyrimidization releases cytosine and uracil, depending on the nucleic acid beingconsidered.