Ribonucleic acid (RNA) Chemical Characterization

Moron, R.S.S., Pazon, A.D., Ramirez, C.V., Raquepo, T.M.R., and Razon, D.N.A, Jr. Recabo,
P.P.L.
2B-Pharmacy, Group No. 6, Department of Pharmacy, Faculty of Pharmacy,
University of Santo Tomas, España Boulevard, 1015 Manila, Philippines

ABSTRACT
The purpose of the experiment is to determine the behavior of Ribonucleic Acid (RNA) towards
various qualitative color reaction tests following alkaline hydrolysis. During Alkaline hydrolysis,
A portion of the isolated RNA was subjected using 2mL 0.3 M NaOH. The RNA hydrolysate was
characterized by different tests: Test for Ribose, Test for Phosphate, Test for Purines (Murexide
Test) and Test for Pyrmidines (Wheeler-Johnson Test). For the test for ribose, the hydrolyzed RNA
yielded a light green solution while the standard ribose solution yielded a dark green solution. For
the test for phosphate the hydrolyzed, unhydrolyzed and standard phosphate solution yielded clear
solutions with no precipitate. For the test for purines, the hydrolyzed, unhydrolyzed and standard
guanine all yielded yellowish-orange residues. For the test for pyrimidines, both the hydrolyzed
and unhydrolyzed RNA both yielded transparent liquids with white precipitate while the standard
cytosine yielded a purple solution.

INTRODUCTION messenger RNA (mRNA) and transfer RNA

Nucleic acids are biomolecules important
for their roles in the storage, transfer and
expression of genetic information. Two
fundamental types of nucleic acids
participate as genetic molecules:
deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). DNA is found
primarily in the chromosomal form in the
cell’s nucleus, where it serves as the
repository of genetic information. [1]
On the other hand, RNA has a wider range
of functions which includes protein
synthesis. RNA is a biologically important
type of molecule that consists of a long chain
of nucleotide units. If DNA is usually double
stranded, RNA is basically a single stranded
nucleic acid. [4] It is usually found at high
concentration in large cytoplasmic volume
due to its specific functions. It is present in
three major types: ribosomal RNA (rRNA),
(tRNA). Each of these three forms plays a
role in the expression of the genetic
information in DNA. Messenger RNA carries
the transient message for protein synthesis
from nuclear DNA to the ribosomes. Transfer
RNA, the smallest nucleic acids, form esters
with specific amino aicds for use in protein
synthesis. It serves as adapter molecules for
the translation of information in mRNA into
a specific sequence of amino acids.
Ribosomal RNA, the most abundant form, is
associated with protein-synthesizing
organelles, the ribosomes. [5] Other classes of
RNA include small nuclear RNA (snRNA),
micro RNA (miRNA) and small interfering
RNA (siRNA) which are also important in
affecting gene expressions.[6]
Nucleic acids are linear polymers
constructed from four different monomers
called nucleotides. Nucleotides have three
characteristic components: a nitrogenous
base, a pentose sugar and a phosphate.[2] All
nitrogenous bases in DNA and RNA are
derivatives of the two heterocyclic
compounds purine and pyrimidine. The
major purines in DNA are adenine and
guanine; the major pyrimidines in DNA are
thymine and cytosine. Similarly, the
predominant purines in RNA are adenine and
guanine; however, the pyrimidines in RNA
are cytosine and uracil. Two types of
aldopentoses are found in nucleic acids.
Ribose occurs in RNA; 2-deoxyribose in
DNA. [3] DNA lacks a hydroxyl group
attached to the pentose ring in the 2’ position
which makes RNA less stable than DNA
because RNA is more prone to hydrolysis. [3]
METHODOLOGY
Reagents used
The materials and reagents used were .3M
NaOH, 10% KOH, Conc. H2SO4, Conc.
HNO3, Ba(OH)2, 10% (NH4)2MoO4 solution,
Br2 water, Orcinol reagent and the standard
solutions namely :ribose, adenine, guanine,
cytosine and uracil.
Procedure
1. Alkaline Hydrolysis
The mixture solution of 2 ml of 0.3N
NaOH and a small amount of isolated RNA
was heated via water bath for 60 minutes. The
hydrolysate was cooled and adjusted to pH 4-
6 with glacial acetic acid using pH paper. The
hydrolysed sample RNA was used for testing.
2. Test for Ribose
A mixture of 0.5 ml hydrolyzed RNA
solution and 2 ml Orcinol reagent was placed
in a water bath for 5-10 minutes. The same
method was used for the standard ribose
solution.
3. Test for Phosphate
1ml of conc. H2SO4 was mixed with the
nucleic acid solutions (e.g. hydrolysed,
unhydrolyzed and standard phosphate). The
tubes were then heated over a small flame
with constant shaking until it went brown.
After cooling, .5ml HNO3 was added and was
heated again until white fumes appeared. 1ml
water was added to the liquid and was heated
for 5 minutes in a boiling water bath and was
then cooled and was again added 1mL 10%
% (NH4)2MoO4 solution. After they were
mixed the tubes are mixed and diluted to 10
mL using water. After letting it stand for 5
minutes the color and precipitate was
observed.
4. Test for Purines (Murexide Test)
5-10 drops of RNA solution (e.g.
hydrolysed, unhydrolyzed and standard
guanine) was placed in a small evaporating
dish. Few drops of concentrated HNO3 were
added to the solution and evaporated to
dryness on a hot plate. The residue formed
was moistened with 10% KOH and heated to
dryness. Few drops of water were added to
the dried solution. Same procedure was done
to the standard guanine solution and
unhydrolyzed RNA solutions. Any change in
color was noted.
5. Test for Pyrimidines (Wheeler-
Johnson Test)
An excess of bromide water was added to
0.5 mL RNA solution until the solution
turned yellow. The solution was boiled on a
 hot plate until a change in color to light
yellow or colorless occurred. An excess of
Ba(OH)2 was added to the solution and tested
with litmus paper. Same methods apply for
the unhydrolyzed RNA and standard
cytosine. Any change in color was noted.

Figure 1. Reaction of ribose with orcinol

reagent .
III. RESULTS AND DISCUSSION

Table 1. Qualitative Colour Reaction Tests 2. Test for Phosphate

results of Standard RNA solution and RNA In the test for the presence of phosphate
Hydrolysate and unhydrolyzed RNA. in both standard phosphate solution and RNA
hydrolysate, a yellow precipitate should be
Test Hydrolyzed Unhydroly Standard
ideally obtained however the group obtained
RNA zed RNA solutions
no ppt. The formation of ppt. is due to the
Ribose Light Green - Dark Green reaction of Ammonium Molybdate solution
soln. soln. which when dropped upon a sample,
indicates the presence of phosphate by a
Phosphate Clear sol, Clear sol, Clear sol, no yellow stain or a crust of yellow phospho-
no ppt. no ppt. ppt. ammonium molybdate. Formation of yellow
crystals follows. However, In this test the
Purines Yellowish- Yellowish Yellowish-
group did not achieve a precipitate at the end.
orange -orange orange
residue residue residue
3. Test for Purines (Murexide Test)
Pyrimidine Clear sol, Clear sol, Clear sol, In the test for purines, or commonly known
s white ppt. white ppt. white ppt. as murexide test, the RNA (both hydrolyzed
and unhydrolyzed) from yeast and the
1. Test for Ribose standard solution yielded a yellowish-orange
Positive results for the standard residue when oxidized with nitric acid and
solution (dark green solution) and for the evaporated due to purine degradation. They
hydrolysed RNA from yeast ( light green turned into yellow residues when moistened
solution) were yielded due to complete with a base, which is a positive result for
conversion of the ribose to an aromatic presence of purine bases and turned back to
aldehyde (furfural) which when reacted yellowish orange ppt (standard solution) and
with Orcinol reagent (3,5-dihydroxy yellowish orange ppt (both hydrolyzed and
toluene) formed an aldehyde-phenol unhydrolyzed RNA from yeast) when water
condensation. was added and evaporated.
Figure 2. Murexide Test

4. Test for Pyrimidines(Wheeler-

Johnson Test)
In the test for pyrimidines, all of the samples
yielded a clear solution with white
precipitate. Ideally, if the sample is treated
with bromine water it should form 5-bromo-
6-hydroxyhydro derivatives which produces
a yellow coloration. Upon dehydration in
solution, it forms a 5-bromo derivative. The
addition of barium hydroxide Ba(OH)2 gives
a 5, 5-dibromo-6-hydroxyhydro derivatives,
a violet precipitate, which is a positive result
for the presence of uracil in RNA. Both the
standard solution and RNA sample (both
hydrolysed and unhydrolysed did not yield 5,
5-dibromo-6-hydroxyhydro derivative
needed to form a violet precipitate when
treated with Ba(OH)2.

A number of samples gave erroneous results.

There is a possibility that contamination
could have occurred in the course experiment
or mainly due to the poor purification method
employed.
REFERENCES

[1] Nelson, D.L. and Cox, M.M. (2000). Lehninger Principles of Biochemistry, 3rd Edition. New
York: Worth Publishers, pp. 325-328, 345-346.

[2] Characterization of Nucleic Acids. Retrieved from

http://www.docshare.com/doc/83332/Characterization-of-Nucleic-Acids 7:17 PM February 8,
2011

[3] Murray, R.K. (1988). Harper’s Biochemistry, 21st ed. Connecticut: Appleton & Lange, pp.
383-386.

[4] Selective Advantage of DNA over RNA as the Genetic Material. Retrieved from
http://www.ncbi.nlm.nih.gov/books/NBK22508/ 8:43 PM February 10, 2011

[5] RNA. Retrieved from

http://virtualology.com/virtualsciencecenter.com/halloforganicchemistry/RNA-
Ribonucleicacid.com/ 6:22 PM February 8, 2011

[6] Meili, M., B. Albert-Fournier, and M. C. Maurel. "Recent Findings in the Modern RNA
World." International Microbiology 4 (2001): 5-11.