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SPECTRAL PROPERTIES OF ACRIDINE ORANGE
BOUND TO DNA/RNA
MO JIANG
ABSTRACT
DNA/RNA, present in almost every cell, has been a hot research topic in a wide range
of areas, including biology, physics, chemistry and polymer engineering. Naturally, to
image DNA and facilitate DNA/RNA-related questions becomes an interesting question
itself.
Since 50 years ago people have been discovering and developing families of
fluorescence dyes (from Acridine orange to YOYO-1) with unique spectral properties
towards facilitating single molecule imaging and operation. Acridine orange (AO) has
been popular since the DNA/RNA research was initiated. AO intercalates into DNA at
low dye/base pair (bp) ratio, showing enhanced green fluorescence, at the same time
absorption spectra red shift and absorption band width narrowing. In contrast, AO
self-associates with RNA or denatured DNA at high dye/bp ratio, showing red
fluorescence. The absorption and emission spectra could be obtained with UV/Visible
spectrometer and spectrofluorometer, respectively.
Even now Acridine orange has still been used in prion disease inhibition and immune
cell DNA/RNA contents quantification, thanks to its affinity to DNA from planar
molecular structure and multi-color fluorescence properties.
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TABLE OF CONTENTS
1. INTRODUCTION....................................................3 1.1 DNA STRUCTURE...................................................3 1.2 DNA BIOCHEMICAL SIGNIFICANCE...................................3 1.3 DNA FLUORESCENCE DYE CLASSIFICATION, PROPERTIES AND GENERAL
APPLICATIONS.........................................................3 1.4 FIGURES AND TABLES...............................................3
2. METHODS AND ANALYSIS..........................................6 2.1 THE INTERACTION BETWEEN AO AND DNA/RNA......................6 2.2 ABSORPTION AND EMISSION SPECTRA OF AO DYE AND WITH DNA/RNA.7 2.3 FIGURES AND TABLES..............................................10
3. APPLICATIONS....................................................14 3.1 INCREASED DNA AND/OR RNA CONTENT OF SYNOVIAL FLUID CELLS IN
RHEUMATOID ARTHRITIS..............................................14 3.2 POTENT INHIBITION OF SCRAPIE PRION REPLICATION IN CULTURED
CELLS BY BIS-ACRIDINES.............................................14 3.3 FIGURES..........................................................15
REFERENCES.........................................................17
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1. INTRODUCTION
1.1 DNA STRUCTURE
The information determining the amino acid sequences in proteins is stored by
long-chain polymeric molecules, the deoxyribonucleic acid (DNA). The most important
feature is that the polymeric chains are twisted about each other in the form of a regular
double helix. Each chain is a polynucleotide, an arrangement of nucleotides in which the
sugar of each nucleotide is linked by a phosphate group to the sugar of the adjacent
nucleotide (Fig. 1). There are 10 nucleotides on each chain every turn of the helix. There
are two pyrimidines, thymine (T) and cytosine (C), and two purines, adenine (A) and
guanine (G). Both types of bases are flat, and they stack above each other perpendicular
to the direction of the helical axis, thus forming the so-called B-helix. The distance of the
stacked bases is 0.34 nm. The two polynucleotide chains are connected by hydrogen
bonds between the bases. Adenine is always paired with thymine (A.T base pair) and
guanine with cytosine (G.C base pair). [1]
1.2 DNA BIOCHEMICAL SIGNIFICANCE
DNA, however, is not the direct template that determines the amino acid sequences of a
protein. These intermediate templates are large polymeric molecules of ribonucleic acid
(RNA), which are chemically related to DNA. The sugar of DNA is deoxyribose, whereas
RNA contains ribose. Moreover, RNA contains the pyrimidine derivative uracil (U)
instead of thymine in DNA. RNA exists to a large extent as a single polynucleotide strand.
Before the amino acids line up against the RNA template (mRNA) they are covalently
attached to relatively small molecules of transfer RNA (tRNA). Each amino acid has its
own tRNA. There is only one way to fold the polynucleotide chain of tRNA under a
maximal number of base pairs: all tRNA form a three dimensional cloverleaf structure.
[1]
1.3 DNA FLUORESCENCE DYE CLASSIFICATION, PROPERTIES AND GENERAL
APPLICATIONS
Fluorescent dyes have become the preferred method of detection for nucleic acids in
molecular biology and biophysics. Some common examples include automated
fluorescent DNA sequencing, fluorescent genotyping, and quantitative target detection
techniques. [2]
The three classes of classic nucleic acid stains (Table. 1) include:
1) Intercalating dyes, such as ethidium bromide and propidium iodide
2) Minor-groove binders, such as DAPI and the Hoechst dyes
3) Miscellaneous nucleic acid stains, including Acridine orange (Fig. 2)
1.4 FIGURES AND TABLES
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Fig.1 DNA and RNA base pair chemical structures [3]
Fig.2 Structure of Acridine orange [2]
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Table.1 — Properties and general applications of classic nucleic acid stains [2]
Dye Name Ex/Em Fluorescence
Emission
Color
Applications
Acridine
orange
500/526
(DNA)
460/650
(RNA)
Green/Red � Permeant
� RNA/DNA discrimination measurements
� Lysosome labeling
� Flow cytometry
� Cell-cycle studies
DAPI 358/461 Blue � Semi-permeant
� AT-selective
� Cell-cycle studies
� Mycoplasma detection
� Chromosome and nuclei counterstain
� Chromosome banding
Ethidium
bromide
518/605 Red � Impermeant
� dsDNA intercalator
� Dead-cell stain
� Chromosome counterstain
� Electrophoresis
� Flow cytometry
� Argon-ion laser excitable
Propidium
iodide
(PI)
530/625 Red � Impermeant
� Dead-cell stain
� Chromosome and nuclear counterstain
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2. METHODS AND ANALYSIS
As a consequence of fluorescent dyes binding to DNA, fluorescence spectroscopic data,
i.e., absorption and emission peak value, band width and fluorescence quantum yield, will
change with respect to the free dye property [1].
2.1 THE INTERACTION BETWEEN AO AND DNA/RNA
The interaction between AO and DNA/RNA depends highly on dye/ base pair ratio (r
value) as in Fig. 3.
At low dye/bp ratio, AO intercalate into ds DNA, so hydrophobic force is important [4]
(Fig. 3, 4)
An accepted model of intercalation [5] characterizes as the following:
1) the total number of possible intercalation sites is fixed a priori to include every slot
between successive DNA base pairs
2) an intercalated dye cation inhibits intercalation at the two slots immediately
adjoining the occupied one
3) an intercalated dye cation may associate with a non-intercalated dye cation to
produce a spectroscopically distinct bound dimer which do not obey Beer’s law . At
degrees of binding between 0.2 and 0.3 mol of dye/mol of DNA phosphate, at least one
additional bound dye specides, presumably of a higher aggregate type, begins to make its
appearance.
At high dye/bp ratio, AO interact with ds DNA by stacking or external binding (Fig. 3),
so ionic binding is important, which sometimes results in DNA denaturation,
condensation and length change. The interaction of AO and denatured ds DNA, could be
treated similar to ss DNA or RNA from chemical structure.[4]
2.1.1 DNA Denaturation
DNA denaturation, also called DNA melting, is the process by which double-stranded
deoxyribonucleic acid unwinds and separates into single-stranded strands through the
breaking of hydrogen bonding between the bases. Intercalation of AO can cause
denaturation of DNA, due to its high affinity for DNA, and co-operativity of binding to
single strand nucleic acid [6].
2.1.2 DNA Condensation
Interaction of cationic dye with nucleic acids often results in condensation of the
product. The driving force of aromatic cation-induced condensation is the cooperative
interaction between dye and single-stranded (ss) DNA. This type of reaction is highly
specific with regard to the primary and secondary structure of DNA, and results in
destabilization of the latter. [7]
2.1.3 DNA Length Change
AO interaction with DNA results in modest changes in DNA dimensions, similar to
yoyo1 [8]. It makes sense as the intercalating dyes reduce the degrees of helix, thus
increase the contour length of ds DNA, although the length of single strand nucleic acid
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remains the same. (Fig. 4)
2.2 ABSORPTION AND EMISSION SPECTRA OF AO DYE AND WITH DNA/RNA
An absorption spectrum shows the fraction of incident electromagnetic radiation
absorbed by the material over a range of frequencies.
An emission spectrum is, in a sense, the opposite of an absorption spectrum. [9]
The absorption bands in AO solutions have been interpreted by Zanker as the result of
the transition from the zero vibrational level of the ground state to the zero, the first and
the second vibrational level of the excitated electronic state, and the emission bands as the
transitions from the zero vibrational level of the excited electronic state to the zero, the
first and the second vibrational level of the ground state. [10]
This is in good agreement with the obvious symmetry in the position and relative
intensity of the absorption and emission bands (Fig. 5), especially for DNA-AO complex.
The increased probability of transitions from the ground state to higher vibrational levels
of the excited electronic state (expressed by intensified short wave absorption bands) is
accompanied by an increased probability of transition from the excited electronic state to
higher vibrational levels of the ground state (as indicated by intensified long wave
emission bands). [11]
For RNA-AO complex, however, this symmetry is somewhat disturbed, since an
additional emission band appears at 15000 cm-1, with no distinct equivalent on the
absorption side. Though hard to explain by direct transition from the excited state to the
ground state, the occurrence of such long wave emission bands indicates an additional
loss of vibrational energy. This can arise if the emission process involves a radiationless
transition from the excited state over an intermediated metastable state to the ground state.
And the demonstration of existence of metastable states involves emission light
polarization, which I do not have enough space to talk about this time. [11]
2.2.1 Absorption Spectra Shift of AO-DNA Complex
Table.2 shows that attachment of AO to ds DNA results in a red shift of the absorption
spectrum of AO, and narrowing the absorption band width. [1]
2.2.1.1 Absorption band width narrowing
The narrowing is due to changes in the vibrational structure of the band, which can also
produce red shift of maximum. [1]
2.2.1.2 Absorption spectra red shift
1) Electro static interaction
On the basis of quantum chemical calculations, salt-like bonds between the negatively
charged nucleic acid-phosphates have been discussed as being responsible for the shift.
[12]
2) Interaction between the heterocyclic ring system of the AO with the nucleic acid
bases
Aπ -electron overlap favors the hypochromicity of the long-wave absorption band of
AO [13]. Quantum chemical calculations performed for aromatic hydrocarbon—DNA
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complexes yielded an increased polarizability of the ligands in the first excited singlet
state [14]. This causes an increase of dispersion forces and a lowering of the excited state
level, a fact possibly responsible for the long-wave shift. In agreement with this idea are
those findings which show that polarization forces contribute to the stability of the
intercalated complexes in the ground state [15] and which may be enhanced in the excited
state.
2.2.1.3 UV/visible absorption spectrophotometer
An UV/Visible absorption spectrometer records the wavelengths at which absorption
occurs, together with the degree of absorption at each wavelength, which forms
absorption spectra. [16] The functioning of a typical spectrometer (Fig. 6) is as follows. A
beam of light from a visible and/or UV light source (colored red) is separated into its
component wavelengths by a prism or diffraction grating. Each monochromatic (single
wavelength) beam in turn is split into two equal intensity beams by a half-mirrored device.
One beam, the sample beam (colored magenta), passes through a small transparent
container (cuvette) containing a solution of the compound being studied in a transparent
solvent. The other beam, the reference (colored blue), passes through an identical cuvette
containing only the solvent. The intensities of these light beams are then measured by
electronic detectors and compared. The intensity of the reference beam, which should
have suffered little or no light absorption, is defined as I0. The intensity of the sample
beam is defined as I. Over a short period of time, the spectrometer automatically scans all
the component wavelengths in the manner described. The ultraviolet (UV) region scanned
is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm. [16]
If the sample compound does not absorb light of a given wavelength, I = I0. However,
if the sample compound absorbs light then I is less than I0, and this difference may be
plotted on a graph versus wavelength, as shown on the right. Absorption may be
presented as transmittance (T = I/I0) or absorbance (A= log I0/I). If no absorption has
occurred, T = 1.0 and A= 0. Most spectrometers display absorbance on the vertical axis,
and the commonly observed range is from 0 (100% transmittance) to 2 (1%
transmittance). The wavelength of maximum absorbance is a characteristic value. [16]
2.2.2 Emission Spectra Properties of AO-DNA/RNA Complex
2.2.2.1 Green/red fluorescence of AO-DNA/RNA
Monomers of acridine orange bind to double-stranded DNA and fluoresce green (-530
nm), whereas polymeric complexes with single-stranded DNA and RNA fluoresce red
(>600 nm).[1]
As mentioned above, at low dye/bp ratio, AO binds to ds DNA by intercalation
manifesting green fluorescence, and this intercalation process stabilizes the double strand
structure against thermal denaturation [11]. At high dye/bp ratio and high AO
concentration (about 100 uM) the red luminescence of the complexes becomes apparent.
According to the classical model [17, 18] the red luminescence of the AO complex with
DNA comes from stacking of AO molecules attached electrostatically to the phosphates
of the ds polymer. [6] The RNA-AO emission is rather similar to that of denatured DNA,
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although the emission peak is shifted slightly.[11]
Besides, it was found that the complex of AO with ss RNA emits a weaker red
fluorescence around 650 nm than the complex with ss DNA at high dye/bp ratios. The
fact could be explained neither by a direct interaction of bound dyes with the 2'-hydroxyl
group of ribose in RNA nor by the difference in the G-C content of the nucleic acids. On
the basis of the character of dye molecules emitting the red fluorescence, it was suggested
that the bases in ss RNA might be buried in some hydrophobic environment that would
make the dyes less likely to interact with them, compared with the bases in ss DNA. [19]
Under strictly controlled conditions when DNA forms an intercalative complex with
AO, while all cellular RNA forms a condensed complex with the dye, the green and red
luminescence is closely related to DNA and RNA concentration in situ. [6] These make
acridine orange a potential candidate for cell physiological study including information
about the composition and structure of DNA.
2.2.2.2 Fluorescence enhancement and quenching
AO green fluorescence is enhanced when bound to DNA at low dye/bp ratio, but
quenched at slightly higher dye/bp ratio. [20]
The increase in fluorescence intensity after intercalation is probably correlated with
rigidity and/or atomic compactness in AO. [21]
From a quantitative point of view, fluorescence enhancement could be reached by
increase of both absorption of AO when bound to DNA with respect to free dye (Fig. 7)
and fluorescence quantum yield (Fig. 8).
The main contribution of absorption comes from the intercalated monomers at low
dye/bp ratio. [10]
The fluorescence quantum yield is the ratio of the amount of output (fluorescence) to
the amount of input (absorption).The numerator is proportional to the total
fluorescence signal coming out of the sample and the denominator is proportional to
the total absorption of the sample. [22] The quantum yield of the unknown sample is
calculated using:
2
2
RR
R R
ODI nQ Q
I OD n=
where Q is the quantum yield, I is the integrated intensity, n is the refractive index,
and OD is the optical density. The subscript R refers to the reference fluorophore of
known quantum yield usually the dye [23]
At low binding quenching is usually caused by the dimer formation at dye/bp ratio
between 0.1 to 0.25, rather than the energy transfer to other monomer molecules which
results in reemission with depolarization. [10]
2.2.2.3 Spectrofluorometer
The fluorescence emission spectra can be obtained by spectrofluorometer (Fig. 9).
Spectrofluorometer is an instrument which takes advantage of fluorescent properties of
some compounds in order to provide information regarding their concentration and
chemical environment in a sample. A certain excitation wavelength is selected, and the
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emission spectrum is recorded. Generally spectrofluorometers use high intensity light
sources to bombard a sample with as many photons as possible. This allows for the
maximum number of molecules to be in the excited state at any one point in time. The
light is either passed through a filter or Monochromator which allows you to select a
wavelength of interest to use as the exciting light. The emission is collected at 90 degrees
to the exciting light. The emission too is either passed through a filter or a
monochromater before being detected by a PMT, photodiode, or CCD detector.[23]
2.3 FIGURES AND TABLES
Fig.3 Proposed structural scheme for the binding of AO to double-stranded (ds) DNA at
various r values [10]
r<0.15, intercalation, dye between adjacent base pairs.
0.15<r<0.30, second dye binds to the exposed part of a dye molecule already partially
intercalated
r>0.30, outside stacking binding without base specificity in addition to second dye binds
to the exposed part of a dye molecule already partially intercalated
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Fig. 4 Acridine orange intercalation into DNA and increases DNA contour length [24]
Fig. 5 Schematic representation of the position and the relative intensitites of absorption
and emission bands of DNA-AO, RNA-AO, and Poly U-AO complexes [11]
Table.2 Acridine orange absorption, excitation and emission spectra peak at different
nucleic acid environment,(ss, single-stranded) [7]
Abs
Max,
nm
Abs
E*10-4
M-1cm-1
Abs
Half
width,
nm
Exi
Max,
nm
Exi
Half
width,
nm
Emi
Max,
nm
Emi
Half
width,
nm
Relative
Emi
Quantum
yield
AO 492 6.85 48 493 46 525 52 1.0
AO+ssDNA
(dye/bp=1.0)
457 4.76 458 630 116 0.2
AO+dsDNA
(dye/bp~0)
502 5.85 40 502 42 522 37 2.2
12
Fig. 6 Beam path of UV/Visible spectrometer [16]
Fig. 7 Absorption spectra of acridine orange-native DNA complexes in 0.001M NaCl
at various values of the binding ratio r (indicated on each curve, 0 stands for free dye).
Total dye concentration 10-6M. [10]
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Fig. 8 Ratio of the quantum yields of fluorescence of DNA-acridine orange complexes
(φ ) to that of the free dye ( 0φ ) as a function of the binding ratio r. Native DNA:
in 0.001M NaCl, 10-5M acridine orange (open circle), 10-6M acridine orange (∆ ); in
0.1M NaCl,10-5M acridine orange (∇ ),10-6M acridine orange (solid triangle).
Denaturated DNA, in 0.001M NaCl, 10-6M acridine orange (solid circle). [10]
Fig. 9 Photon-counting spectrofluorometer from ISS. [22, 23]
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3. APPLICATIONS
3.1 INCREASED DNA AND/OR RNA CONTENT OF SYNOVIAL FLUID CELLS IN
RHEUMATOID ARTHRITIS
Numerous studies have provided evidence which suggests a major role of
cell-mediated immune responses in the pathogenesis of rheumatoid arthritis (RA).
Following stimulation, T cells may become activated and proliferate. [25]
To determine the respective proportions of proliferating and activated cells
(characterised by their increased DNA and RNA contents, respectively) in synovial fluids
from patients presenting with RA of various durations and stages of activity,
flow-cytometry studies were carried out in acridine orange-stained synovial fluid
lymphocytes.
Fluorescence stained cells were run in a 50H Cytofluorograf (Ortho Instruments,
Westwood, USA) to provide simultaneous measurements of the scattered light and the
green fluorescence of each cell, as it passed through the 488 nm line of an argon-ion laser
(total power = 5 watts). Simultaneous measurements of the forward angle scatter (FAS,
proportional to cell diameter) and the right-angle scatter (RAS, proportional to cell
volume and protein content) were used to identify synovial fluid leucocytes. Green and
red emitted fluorescence were simultaneously measured for each cell as it passed through
the focused laser. From the cytogram we could determine the distribution of cells within
the phases of the cell cycle.
As in Fig.10, the DNA of the total lymphocytes from synovial fluid displayed the same
distribution (Fig. 10 2A) as that of normal peripheral blood lymphocytes with a high peak
corresponding to the G0/G1 phase. The second peak corresponded to cells in the G2 + M
phase. The part of the histogram between these two phases corresponded to cells
progressing through the S phase. The shape of RNA distribution histograms was either
bimodal (Fig. 10 2B), as reported for lymphocytes from normal blood, or unimodal (Fig.
10 2C). Lymphocyte activation causes the RNA cell content to increase, as shown by a
shift to the right on the histogram.
Together with biochemical measurements of membrane specific immune markers, they
came to the conclusion that changes in cellular DNA and/or RNA contents provide a
valuable parameter of lymphocyte activation, although not necessarily linked to the
expression of differentiation antigens by activated cells. [26]
3.2 POTENT INHIBITION OF SCRAPIE PRION REPLICATION IN CULTURED
CELLS BY BIS-ACRIDINES
Prion diseases in humans and animals are invariably fatal. Prions are caused by
stimulating the conversion of the normal host prion protein (PrPC) into disease-causing
isoform (PrPSc). The conversion is not clearly understood at either a molecular or cellular
level, thus compounds that exert a dramatic effect on PrPSc concentrations, such as
bisacridines (Fig. 11), can serve as tools to probe prion biology.
Fig. 11 shows one of such compounds. It was proven to reduce PrPSc levels in ScN2a
cells in a dose dependent manner (Fig. 12), without affecting PrPC. ScGT1 cells could
also be cured of PrPSc after 1-wk incubation with bisacridine in Fig. 11. What is more,
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treatment with bisacridine appears to decrease the capacity of ScGT1 cells to be
reinfected with prions, relative to controls.
The possible mechanism of bisacridine’s effect on prion inhibition is unknown yet. It is
possible that the planar structure of acridine with high affinity for DNA blocks DNA
replication and the following protein translation process. In addition, the linker
dependence observed for the polyamine series (change the linker length of bisacridine
compound in Fig. 11) hints that both acridine heterocycles can occupy independent
binding sites to affect PrPSc load in scrapied cells. [27]
However, considering the high affinity of acridine to DNA, the challenge in targeting
bis-acridine-based therapies is to separate the desirable bioactivity of these compounds
from their DNA bisintercalative cytotoxicity. To achieve a balance between desirable
bioactivity and cytotoxicity, people are trying to target sterically constrained bis-acridine
analogs. Intercalation of bis-acridines can be mitigated by using rigidified or sterically
constrained linkers to tether the acridine heterocycles [28]
3.3 FIGURES
Fig. 10 Distribution histograms of DNA (2A) and RNA contents (2B and 2C) from
different cell suspensions. [26]
16
Fig. 11 Bis-acridine compound, nanomolar inhibitior of PrPSc replication [27]
Fig. 12 Dose response for bis-acridine compound inhibiting of PrPSc replication in ScN2a
cells by ELISA (squares, lower curver) and Western blot densitometry (diamonds, upper
curve) [27]
Fig. 13 The observed bioactivity of polyamine-linked bis-acridine in ScN2a cells
correlates to the length of the polyamine linker [27]
17
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