1

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.

2

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

3

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

4

Fig.1 DNA and RNA base pair chemical structures [3]

Fig.2 Structure of Acridine orange [2]

5

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

6

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

7

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

8

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,

9

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

10

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

11

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]

13

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]

14

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,

15

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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