detailed literature review of G-quadruplex ligand binding assays
3rd yr M.Pharm project
Al-Nasir & Owez Madhani
Kingston School of Pharmacy and Chemistry
& St. Georges Hospital Medical School
are secondary nucleic acid structures formed in guanine rich regions.They
comprise of stacks of square-planar hoogsteen bonded guanine nucleotides known
G-tetrads. G-Quadruplex conformations can be found to form in telomeric and
oncogene regions and are implicated in the expression of growth factors. Hence
they are potential targets for therapeutic agents. The search for these ligands
that bind and stabilise
G-Quadruplexes is therefore an important one. To assess if these ligands are
ideal drug candidates various ligand binding assays may be carried out. Such
assays yield information on binding affinity, selectivity, stoichiometry, and
conformation for ligand and G-Qudruplex interactions. In this literature we
outline the fundamentals of SPR, PCR-stop, ESI-MS, Circular dichroism, FRET,
TRAP, G4-FID and TRE assays with respect to G-Quadruplex ligand binding.
type structures consists of Guanine rich nucleic acid first discovered by Davies
et al in 1962.1 In this paper we will look at significance of this
type of structure and discuss various assays used to study its’ ligand binding
potential. According to Dr Julian Huppert a
Computational biologist at Cambridge university, “Nucleic
acids are capable of forming a wide variety of different structures, far removed
from the Watson-Crick double helix…Many of the alternative structures that can
be formed have physiological functions, such as controlling gene expression via
gene transcription or translation.” (Huppert, 2006). G-Quadruplexes are such structures and their importance is implicated
in a variety of intracellular processes such as gene expression, telomerase
regulation and signal transduction.
to the G-Quadruplex structure
G-Quadruplex (i.e. Guanine-Quadruplex) type structure consists of a
four-stranded nucleic acid formed from guanine nucleotides. As we shall see
later G-Quadruplexes can form from a single strand or from multiple strands. A
repeating motif in this four-stranded structure is the
also known as a Tetrad, which is a square, aromatic plane of four
guanine nucleotides held together by Hoogsteen bonding.
– G-Quartet (tetrad)
are often stacked on top of one another to form the G-Quadruplex
structure itself. It is under appropriate
conditions (high Na+ or K+ ionic strength) that
segments of DNA or RNA that is rich in Guanine residues can fold
to form G-Quadruplex structures and is known as G4-DNA and G4-RNA
The constituent tetrads stacked on top of each other can co-ordinate metal
cations such as Na+ and K+ as each pair of tetrads
contains eight oxygen atoms. It has been shown that G-Quadruplexes exhibit
conformational polymorphism that is influenced by the polarity of the parallel
(Figure 1.1) - Schematic showing the fold in the intermolecular
quadruplex formed by two molecules of d(GGGGTTTTGGGG)
(Figure 1.2) – Cartoon of possible G-Quadruplex structures
adopting intermolecular and intramolecular configurations.
1.2 above depicts possible types of G-Quadruplex structure, intermolecular
G4-DNA comprised of different strands (A and C), and intramolecular G4-DNA where
the bonding occurs on the within the same strand (B).
contrast to Watson-Crick bonding which involves N1 and N3 of the heterocyclic
rings, Hoogsteen bonding involves N7, and occurs between this N7 and N3 on the
corresponding nucleotide. In Hoogsteen bonding the purine is in a syn
comformation as opposed to anti in Watson-Crick base-pairing. The Hoogsteen base
pairing scheme is involved in stabilising triple stranded DNA, and is of
interest to us as it also stabilises the Q-Quartet.
- Watson-Crick Base pairing
- Hoogsteen Base pairing
DNA: Potential therapeutic targets
are significant as there are numerous guanine rich regions in the genome that
are involved in regulatory functions. For instance telomeres located at the
terminal ends of chromosomes are non-coding portions of DNA rich in Guanine
where G-Quadruplexes form. These telomeric regions of DNA shorten on each
replication. However the number of successful replications before the telomeres
are depleted, and the cell cycle stops is finite, and is known as the Hayflick
limit. Telomerase counteracts this senescence (ageing) process by repairing the
telomeres, by adding repeats of TTAGGG. This operation increases the number of
divisions the DNA may take part in. In normal cells telomerase expression and
hence activity is low and thus limits the life-span of the cell’s line..
However in cancerous cells, where DNA replication is uncontrolled, telomerase
expression and activity are increased. As telomeres are rich in Guanin, G-Quadruplexes
form and these are potential targets for ligands that bind the Quadruplexes and
interfere with telomerase activity thereby reducing tumour growth.
G-Quadruplex structure as a therapeutic target in the treatment of cancer is an
exciting possibility given the extreme cytotoxicity and un-specificity of
traditional alkylating anti-cancer agents. 
telomeres contain various conformations of G-Quadruplex nucleic acid and this is
of consequence to ligand binding. Conformation is related to environmental
conditions and 125I-radioprobe studies reveal chair and propeller
conformations in K+ solution whilst NMR studies show that basket and
anti-parallel basket type conformations predominate in Na+ solution.
The basket conformation exists in both ionic solutions. Since intracellular K+
is general high compared to that of Na+ studies of Q-Quadruplex
conformation K+ is therefore thought to be of more biological
are genes, which if sufficiently expressed, change a normal cell into a
cancerous one in which apoptosis (pre-programmed cell death) does not occur and
DNA replication continues uncontrollably.
c-myc oncogene contains a region known as NHE III (nuclear hypersensitivity
agent) which responsible for controlling 85-90% of transcription activation.
Stabilising G-Quadruplexes in this region with ligands has the effect of
inhibiting transcription and therefore decreasing c-myc expression.
such as bcl-2 are precursors to oncogenes, which in their normal state or their
normal expressed level constitute part of the normal cell cycle and are not
oncogenic. However, when altered or over expressed they result in a cancerous
state of altered senescence or inhibited apoptosis which constitutes a lack of
control over DNA replication. The oncogene Bcl-2 is yet another guanine rich
region and is capable of forming a mixture of G-Quadruplex structures which are
potential therapeutic targets. 
(vascular endothelial growth factor) is involved in the angiogenesis of tumour
growth, that is the vasculisation that supports tumour growth. This is another
potential therapeutic target as the formation of G-Quadruplexes are implicated
in VEGF expression. 
ligand binding assays
that the potential clinical significance of G-Quadruplexes has been established
it is logical to study the interactions between this type of structure and
it’s ligands. There are a variety of methods available, some utilise a range
of routine analytical techniques, others involve more specific protocols.
techniques: ITC (Isothermal titration calorimetry)
and DSC (Differential scanning calorimetry)
techniques such as ITC and DSC provide a means of quantifying the thermodynamic
properties and processes of G-Quadruplex ligand systems. The characteristic
conformational polymorphism of G-Quadruplexes, which may alter on binding or
un-binding is a physico-chemical process that can be monitored by DSC. For
instance in DSC a sample of a G-Quadruplex-ligand complex is heated and any
resulting conformational change results in a corresponding change in specific
heat of the system, ΔCp which is measured and provides useful quantifiable
data can be obtained from DSC because an equilibrium exists between different G-Quadruplex
conformations or G-Quadruplex-ligand complexes and stability is proportional to
the amount of heat required to alter the conformation. The transition mid-point,
Tm is the temperature at which the equilibrium is a 1:1 mix of each
DSC monitors the temperature changes of a single analyte as a whole (i.e. the G-Quadruplex-ligand
complex), ITC works by introducing the two binding species, the ligand and the
G-Quadruplex separately. On molecular binding of the species, heat is either
absorbed or released and this is measured to provide detailed thermodynamic data
in the form of an Isotherm which can be used to calculate various parameters
such as binding enthalpy, Kb, binding stoichiometry, Gibbs free
energy, and entropy change.
Chain Reaction assays
Polymerase chain reaction, is a pioneering method used
to synthesise DNA without the requirement for bacterial cells. (Karp cell
biology, 2004) Developed in 1983 by Nobel laureate Kary Mullis, PCR is based
on a similar polymerase reaction first proposed by Nobel laureate H.
Gobind Khorana and Kjell Kleppe in 1967. Both methods involve a polymerase
enzyme that generates DNA from single stranded oligonucleotide primers which are
complementary to the DNA that is to be synthesised. PCR utilises Taq polymerase
isolated from Thermus Aquaticus bacteria (a thermophilic species), and uses
thermal cycling is used to “melt” the DNA into complementary single strands
that are again replicated by the polymerase. Thermal cycling followed by the
polymerase reaction results in an exponential production of DNA, and hence it is
known as an “amplification” type procedure.
The PCR process consists of the following stages: -
are numerous derivations of the polymerase chain reaction, and one assay that is
of particular significance to us is the PCR stop assay. PCR stop is used to
study interference of ligands on polymerase activity. PCR stop assays are
therefore an important means of assessing the binding of stabilising ligands on
G-Quadruplex DNA. If bands of paused polymerase activity are found at guanine
rich segments of electrophoresis assays on PCR products this indicates efficacy
of ligand stabilisation, and the intensity of these paused bands is proportional
to the inhibition of polymerase activity.
of a PCR stop assay
reaction mixture consisting of Template DNA and primers (Texas red labelled
primer) were heated to 95 C for 3 minutes in a reaction buffer (10mmol Tris-HCl,
pH 8.30, 50mmol KCl, 1.5mmol MgCl2). This was then allowed to cool for 30
minutes. The ligand to be assayed was added and the reaction mixture incubated
at ambient temperature for 1 hr. Taq polymerase was then added together with
dNTPs and incubated at 48 for 30 minutes. Reaction products were then sequenced
using an automatic sequencer. (ligand tandem)
(Telomere Repeat Amplification Protocol) assay
assay utilises PCR and quantifies the activity of telomerase, which is directly
proportional to the amount of TTAGGG repeats added to the telomere. TRAP uses
two primers, M2 (also known as Telomerase substrate) which is the forward primer
required for telomerase addition of TTAAGGG repeats, and CX which is the reverse
M2 (TS) Primer:
5’-AAT CCG TCG AGC AGA GTT-3’
5’- CCC TTA CCC TTA CCC TTA CCC TAA-3’
annealing of the repeats occurs at 25°C
and PCR is then used to amplify the products. The resultant products are then
electrophoresed using PAGE (Polyacrylamide gel). (childrens medical research
of a trap assay
primer M2 (50ng/ μl)
primer CX (50ng/ μl)
polymerase (5U/ μl)
reaction mixture according to above specifications.
μl into thin-walled PCR tubes.
μl of cell or tissue extract (1 μg/ μl) per tube.
telomerase reaction to proceed for 30 minutes at R.T.P.
with oil and place the tubes in PCR machine and run following thermal cycling
PCR products are then electrophoresed on Sybergreen stained Polyacrylamide gel.
(Surface plasmon resonance) assay
a type of photoelectric analysis technique exploiting surface plasmons,
electromagnetic waves that occur near the surface of an adsorptive surface
(~300nm). These waves alter the refractive index of the solution near the sensor
and the extent of this modulation is directly proportional to the number of
molecules within the vicinity. One of the ligands is immobilised at the sensor
surface whilst the analyte is injected, as the analyte binds to the ligand the
concentration of molecules increases around the sensor, thereby altering the
refractive index. SPR is a fast and sensitive technique useful in screening
libraries of small ligands and is ideally used to characterise interactions
between ligands and macromolecules such as
equipment generates a plot of response vs time known as a sensorgram and another
corresponding sensorgram is created for background activity. The sensorgram of
background activity is then subtracted from the first in order to yield a
measure of the effect of the binding interaction on the response (the change in
refractive index). A sensorgram usually has a characteristic increase in
response as the species bind known as the association phase and a corresponding
decrease, the dissociation phase when the species depart from the sensor
The unit of measurement for Response is the aptly named Response Unit, RU and a
single RU is equivalent to the binding of 1pg of protein per mm2 of
is able to analyse equilibrium measurements such as binding affinity and
enthalpy. G-Quadruplex-ligand interaction affinity constants determined by SPR
also generally correlate well with other methods such as Fluorescent titration,
TRAP and thermal melting studies. However equilibrium analysis requires multiple
and sequential injections of analyte at different concentrations, and this
limits its practical use to ligands that establish equilibrium within around 30
minutes. Analytes having a low dissociation constant (KD < 10 nM)
are highly affinitive and have very slow rates of dissociation, Koff and are
therefore unsuitable for analysis. The use of SPR for equilibrium analysis is
thus practically restricted to those ligands with a high dissociation constant (KD
provides data on the stoichiometry of the G-Quadruplex-ligand interaction.
According to Redman, "The maximum response observed when all surface
binding sites are saturated is proportional to the mass of bound analyte, which
in turn is proportional to the molecular weight of the analyte, the number of
binding sites per immobilized ligand, and the surface density of the ligand. The
expected maximum response, RUmax, for every molecule of analyte bound
per molecule of ligand can be calculated from the response obtained during
loading of the chip with ligand, and the molecular weights of the ligand and
designed to bind with Quadruplex DNA are often cationic and aromatic so as to
stack with the terminal Tetrad segments of the Quadruplex. These properties post
problems for SPR in that the solubility of aromatic compounds in aqueous
solutions is problematic and the SPR sensor requires a predominantly aqueous
solution. However, the use of water imiscible co-solvents such as DMSO in small
amounts (as much as 1%) is often employed to overcome the issue.
of Quadruplex structures that have been immobilised onto SPR sensors
ESI-MS (Electrospray mass
ionisation mass spectrometry is a powerful analytical tool with numerous
applications. Whilst a detailed discussion of its applications are beyond the
scope of this paper, it suffices to say that the technique is a “soft” form
of mass spectrometry in which very little fragmentation occurs and non-covalent
interactions are preserved in the process. This is of particular biological
significance and facilitates the analysis of proteins where it is desirable to
preserve these non-covalent interactions within the gas phase.
ESI-MS, the analyte is very slowly injected into the apparatus by means of a
syringe driver at a rate of around 1µl/min. The solution passes through a
needle and plate between which there is a large potential difference that
accelerates the sample. Solvent evaporation of the sample occurs and the
droplets break apart, a process termed couloumbic explosion, when their surface
tension cannot support their charge (known as the Rayleigh limit). The particles
then usually possess multiple charges distributed around different sites of the
protein and enter the mass spectrometer for separation according to m/z ratio.
ESI-MS uses a minimal amount of sample, is a fast technique and yields the
stoichiometry of the interaction making it very useful tool for bimolecular
analysis between two species.[bristol]
of an ESI-MS assay 
binding affinity and stoichiometry of three ligands (depicted below) to G-Quadruplex
DNA of the human telomeric sequence AGGGTT was assessed investigated. The buffer
used for the procedure was NH4Oac.
and apparatus settings
Finnigan LCQ XP Plus ion mass trap spectrometer
100 C with a double sheath gas
of the G-Quadruplex
(Figure 3.0) – Top: G-Quadruplex Tetrad, Right: A selection of
3 ligands studied.
ImImImβDp (Ligand A)
Tel01, pyrilene derivative (Ligand B)
PyPyPyγImImImβDp (Ligand C)
Quadruplex was mixed with the three ligands in molar ratios ranging from 1:1 to
1:8. Binding affinity was evaluated by comparing the ratio of abundances of the
[complex] to [Quadruplex DNA].
Base peak at m/z 1514 is the Q5- ion and represents the G-Quadruplex
Spectrograph of Ligand A (ImImImβDp)
three peaks right of the base peak correspond to ratios of complex 1:1,
1:2 and 1:3 and show relative abundances of 73%, 27% and 18%. For
instance [Q+3III] represents the complex + 3 mols of ligand and so
forth, and the binding affinity as previously mentioned is a measure of
the relative abundances of complex to Quadruplex (base peak) at m/z
the spectrograph of the Tel01 ligand the Q5- ion practically
disappeared and so another base peak was chosen.
The disappearance of the Q5- ion is indicates very good
binding of the Tel01 ligand to the G-Quadruplex DNA. This was
additionally confirmed by the decrease in Q5- ion peak with
an increase in ratio of Ligand:Q-Quadruplex-DNA.
Spectrograph of Ligand B (Tel01)
Spectrograph of Ligand C (PyPyPyγImImImβDp)
ligand C the spectrograph shows a base peak for the Q5- ion
at it’s highest and no peak for the complex ion was observed.
These observations indicate the lack of binding of the ImImImβDp
ligand to the
results of the ESI-MS assay indicate the preferential binding of the ligands to
G-Quadruplex DNA to be Tel01 > ImImImβDp > PyPyPyγImImImβDp.
dichroism utiliseses plane polarised light that is rotated about it’s axis to
create a helical light wave from the source. The ability of chiral of
stereocenters is exploited and measured as a change in Molar Ellipticity from
which structural and conformation information about the analyte can be deduced.
CD is a useful tool for characterising G-Quadruplex structures and can provide
information on binding stoichiometry of G-Quadruplex-ligand interactions.
Circular dichroism depicts the changes in conformation that such ligand
interactions may cause to the G-Qudruplex structure.
of a CD assay utilising Papaverine derivative ligands 
5.0) - Papaverine derived ligands
uM of oligonucleotide was placed in a TRAP buffer and the test ligand added in a
drug:quadruplex ratio of 10:1. CD spectra measurements in the range of 220-550nm
were repeated at 5um of G-Quadruplex oligonucleotide. Results were obtained from
averaging three scans with a scan of the buffer solution subtracted.
- Spectra of
G-Quadruplex htel21 (2.5um)
with ligands 1 (spectrograph A) and ligand 2 (spectrograph B).
insets show corresponding plots of change of molar ellipticity with
addition of ligands. (260 or 270nm).
- Spectra of
G-Quadruplex htel22 (2.5um)
with ligands 1 (spectrograph A) and ligand 2 (spectrograph B).
conditions as above).
- Spectra of G-Quadruplex htel24
(2.5um) with ligands 1 (spectrograph A) and ligand 2 (spectrograph B).
conditions as above).
Titration experiments were also carried out in TRAP buffer. The spectra recorded
of the oligonucleotides before ligands were introduced is characteristic G-Quadruplex
in K+ conditionsm and this is depicted by a “distintive shoulder” at ~270nm
and a smaller negative peak at ~230nm. The relative intensities of each peak
differs between each of the oligonucleotides, however the overal pattern is the
of the ligands to the oligonucleotides results in conformational change
confirmed by an alteration to the CD spectra. For ligand 1, the peak at 250nm
disappeared whereas for ligand 2 this same peak was preserved. The major
positive band (the distinctive shoulder) near 290 showed an increase in molar
ellipticity. The alterations in the spectra of
G-Quadruplexes suggest that they are modified by complexation of the ligands.
least two conformations of G-Quadruplex are suggested to occur in K+ solution,
namely the basket-type and anti-parallel. A hybrid form is also likely to occur
and is characterised by a shoulder near 270nm.
of the basket structure was observed with ligand 1 as the 250nm peak
disappeared. Ligand 2 however was found to stabilise the basket structure
as the 250nm peak was preserved.
(Fluorescence Resonance Energy Transfer) melting assay
melting assay can determine the ‘affinity’ and ‘selectivity’ of ligands
by measuring the increase in melting temperature of a quadruplex induced by the
linkage of ligands to G4 DNA.
Principle of FRET:
is a fluorescence-based spectroscopic method which provides distance based
information on structural changes of macromolecules 13.
A typical FRET experiment involves covalently attaching a polymer with two
fluorophore probes, a donor and an acceptor. The pair should be such that the
emission spectrum of one probe (donor molecule) coincides with the absorption
spectra of the other (acceptor molecule). In which case, fluorescent energy is
transferred from the donor to the acceptor in a non-radiational manner. This
transfer is only possible when the pair is within a certain distance from each
other. This distance within which the non-radiative energy transfer is possible
is known as the ‘Förster distance’ (R0) and is characteristic
for the donor-acceptor pair. It is usually within 10 - 80 0A 14.
the probes are within this distance the fluorescence emission of the donor is
lower than when they are further apart (because the energy is transferred to the
acceptor). Now when the distance increases such that R > R0, the
energy would no longer be transferred to the acceptor and the energy peak in the
emission spectra of the donor increases accordingly 14.
principle is applied in a ‘FRET melting assay’ where increase in temperature
leads to denaturing (melting) of the macromolecule. Denaturing causes the
distance between the probes to increase leading to an increase in fluorescent
::The FRET melting assay
a FRET melting assay, a G-quadruplex forming oligonucleotide is labelled with a
pair of fluorescent probes (donor-acceptor pair). To explain the working of the
assay we have selected the fluorescent dye ‘fluorescein’ (FAM molecule) as
our donor and ‘tetramethylrhodamine’ (TAMRA) as an acceptor dye and a single
stranded guanine rich nucleotide chain with 21-bases as our quadruplex forming
oligonucletide (QFO). Their structures are shown below:
6.0) Structure of the QFO, donor and the acceptor molecules.
taken from Juskowiak B.14)
FAM and TAMRA are attached to the 5’ and 3’ ends of the
oligonucleotide respectively as shown in the figure below. This FAM-TAMRA
dually labelled oligonucleotide is called ‘F21T’.
6.1) Structure of the dual probe labelled QFO.
FRET system shown above we can measure the distance between the donor (FAM) and
the acceptor (TAMRA) probes in terms of the fluorescence of the donor (FAM).
the theory states, at low temperatures the oligonucleotide is folded in a
quadruplex structure and thus the distance between the probes falls within the Förster
radius (R0). This causes the emission radiation of FAM being quenched
as the temperature is increased closer to the melting point (denaturation) of
the oligonucleotide, it unfoldes and the distance between the probes increases
beyond the förster radius (R0) and the quenching effect of the donor
emission (by the acceptor) decreases. This leads to increased donor emission.
simple representation of the above mentioned theory is shown below:
6.2): Figure at the top shows the emission
spectrum of FAM when the probes are within the Förster radius (shown
here as R0) The figure
at the bottom shows the spectrum after the distance between the probes
has increased beyond R0 (due to unfolding of the quadruplex
structure caused by increase in temperature)
FAM tag is excited at 492 nm, with a 9 nm full width at half
maximum (FWHM) filter, and the emission is collected at 516 nm
(10 nm FWHM filter)]
*(Graphs not to scale)
It can be
seen from the diagram that as the distance increases (or as the temperature,
unfolding of the oligonucleotide increases) the fluorescence energy of the donor
order to quantify the effects of temperature, the fluorescence energy
measurements of the dual probe coupled oligonucleotide are conducted with step
wise increase in temperature.
oligonucleotide (F21T) is allowed to equilibrate at 250C for 5
minutes such that it forms a folded (G-quadruplex state) and the donor emission
energy is at its minimum. The temperature is increased by 10C every
minute to 950C and the fluorescence energy is recorded every minute.
The simple representation of the graph obtained from such an experiment is shown
6.3) Graph on
the left shows the increase in fluorescence with increasing temperature. Graph
on the right is similar although the emission values have mean normalised to the
scale of 0 to 1
is somewhat impractical to calculate a true melting point for macromolecules
like an oligonucleotide. Thus we use T ½ for comparison studies. T ½ is the
temperature value for which the normalised emission is 0.5. It can be seen from
the graph that its value for ‘F21T’ is approximately 530C.
on addition of putative ligands which stabilise the folded G-quadruplex
structure, the melting point (or the denaturation temperature) of the ligand
bound oligonucleotide should increase. Thus the T ½ values should increase.
extent of change in T ½ values (∆ T ½ ) on addition of ligands to F21T
from the values obtained from F21T alone are indicative of stabilisation of the
G-quadruplex structure due to ligand binding. The higher the ∆T ½ value
for a ligand, the better is its affinity.
purpose of reviewing we have selected the following shown ligand:
Structure of the ligand used for the FRET melting assay
modified from Cian A D. et al
conducting controlled melting experiments as demonstrated above with mixtures
F21T and the above shown ligand at different concentration strengths, the
following results are obtained:
6.5) Graph showing
the extent of melting temperatures for F21T due to binding to ligands.
Here the same ligand is used at
different concentration strengths. Strengths moving from left to right
following the curves are 0μM, 0.5μM, 1μM, 2μM, 4μM
lines are from duplicate measurements. (Image
modified from Cian A D. et al
It can be
seen from the graph above that the ligand concentration of 8μM leads to a
∆T ½ of (77 0C – 530C) = 240C.
literature, ligands causing ∆T ½ of >200C at less than 1μM
concentration are the best at stabilising G4 quadruplex. Our selected ligand
(figure 6.4) require more than 3μM to cause ∆T ½ of 200C.
Thus it is not as effective, however, higher concentrations of 8μM as seen
above give 240C stabilisation.
selectivity is an important factor to consider when selecting a ligand as a
potential drug target. FRET melting assays can also show ligand selectivity. It
can be measured by adding a non-tagged (non-fluorescent) duplex DNA into the
test solution containing ligand bound F21T oligonucleotide and then conducting
the melting experiments.
the values obtained from this experiment with the values obtained from ones in
which a competitor DNA is absent, one can measure the extent of ligand trapping
(binding of ligand to the duplex DNA) by measuring the decrease T ½ values.
of FRET melting assays 13, 14, 15:
analysis to be conducted in a variety of ionic conditions.
The method is rapid and convenient.
It can easily be adapted for high-throughput
of FRET melting assays 13, 14, 15:
can only achieve semi-quantitative results for ligand affinities.
quadruplex forming oligonucleotide has to be modified.
method can generate false positives (quenching of the donor emission by the
tested ligand) or false negatives (folding of the QFO with the ligand in
such a configuration that the distance prevents quenching)
(G-quadruplex Fluorescent Intercalator Displacement) assay:
assay is based on the displacement of a fluorescent probe by putative ligands
from DNA matrices. This test measures the affinity by which the ligands bind and
their selectivity over different types of DNA matrices.
procedure measures the loss of fluorescence of the bound probe by its
displacement due to a DNA binding ligand.
For the purpose of describing the workings of this assay, we would select one
ligand analogue and show how we can test its binding affinity to a quadruplex
forming oligonucleotide (QFO) and its selectivity over duplex DNA.
experimental data as presented below has been obtained from Monchaud
D. et al 11.
probe: Thiazole Orange.
This molecule binds to the
quadruplex-forming oligonucleotide 22AG in a single-site manner, with high
affinity (Ka = 3 × 106 M−1).
Its fluorescent quantum yield is very low when free in solution (ΦF = 2 × 10−4),
however it increases by 500-1000 fold on binding to DNA. Hence its
displacement by test ligands can be monitored by the reduction in TO
fluorescence at λmax = 539 nm.
structure: 22AG (a quadruplex
forming oligonucleotide which mimics human telomeric sequence [5′-AG3(T2AG3)3-3′])
in K+ buffer
DNA strand: It is a 17
base pair duplex stranded DNA (ds 17). The sequences of the two
complementary strands are: [5′-CCAGTTCGTAGTAACCC-3′] &
A N-methylated quinacridine
Titration of Thiazole orange (TO) with the solution of 22AG:
is carried out in a 3 ml cell at 200C in a 10nM sodium cacodylate
buffer pH 7.3 and 100mM KCl. The addition of TO is followed by 22AG at 501nm
excitation wavelength. Data is then collected by scanning a range of wavelengths
of UV-Vis light from 510 to 750 nm. The graph obtained by plotting fluorescent
intensity against wavelength (510 nm – 750 nm) would be similar to the one
depicted in figure 1 below. The area under curve is FA0 which is
known as the Fluorescent Area; when no ligand exists in the test solution.
G4-FID assay (addition of MMQ16 to 22AG):
similar experimental conditions as above (i.e. 3ml cell, 200C
temperature, buffer solution and KCl), 0.25 µM pre-folded 22AG is mixed with
0.50 µM TO. The ligand MMQ16 (different concentrations) is then added to the
solution. After a 3 minute equilibrium period a fluorescence spectrum is
obtained similar to figure 7.0 below. However, in this case the area under curve
obtained (or the Fluorescent area, FA) is reduced due to the displacement of TO
by the ligand MMQ16.
7.0). Fluorescent spectrum of TO bound to 22AG obtained by
scanning UV-Vis wavelengths from 510nm to 750nm.
scan shown is not up to scale and shows the area under curve or the
fluorescent area (FA0, blackened) before the addition of
the ligand MMQ16.
Fluorescent spectrum of TO bound to 22AG obtained by scanning UV-Vis
wavelengths from 510nm to 750nm.
scan shown is not up to scale and shows the reduction in the area
under curve or the fluorescent area (FA, blackened) after the addition
of the ligand MMQ16.
can similarly be obtained for ds 17 (Duplex DNA) following the above procedures.
of the results:
the results obtained from the assay, the percentage displacement of TO by the
ligand at different ligand concentrations can be calculated as follows:
% TO displacement values can be plotted against ligand concentrations (µM) to
obtain a graph as follows:
Graph showing the % TO displacement from 22AG in K+ and ds
17 at different MMQ16 concentrations.
conditions: [oligonucleotide] = 0.25 μM, [TO] = 0.5 μM
for 22AG and TBA, 0.75 μM for ds26, cacodylate buffer.
modified from Monchaud et al.11)
of a ligand for the DNA matrix (22AG or ds 17) can be obtained as a
concentration value at which the ligand leads to 50% of TO displacement. From
the graph (Fig. 7.2), the affinity values for MMQ16 can be obtained as follows:
to G-quadruplex forming 22AG is the concentration of MMQ16 which causes 50%
of TO to displaced from the binding sites on the quadruplex.
The value from the graph is G4DC50 (22AG K+) = 0.14 µM.
Affinity to duplex DNA (ds
17) can be similarly obtained from the graph.
Which in this case is dsDC50 (ds 17) > 2.5 µM (as it only causes 43.2%
Selectivity of MMQ16 for G-quadruplex
DNA over duplex DNA can be obtained by the ratio of dsDC50 / G4DC50 values.
from above, a G4-FID assay can evaluate a ligand’s affinity and selectivity to
G-quadruplex DNA over duplex DNA. Affinity values show how strongly a ligand
binds to the DNA whilst selectivity values show a ligand’s tendency to bind to
G-quadruplex DNA over duplex DNA.
test doesn’t require modified oligonucleotides. Thus a variety of DNA
matrices (different G-quadruplex conformations, duplex DNA etc.) can be
specific requirements (Compounds used are readily available and equipments
generally include a standard spectrofluorimeter for fluorescence
measurements and a standard spectrophotometer for UV-Vis measurements) 
materials used in the assay are inexpensive and the procedure is neither
technically demanding nor time consuming [11,12]
broad diversity of ligands can be evaluated by this assay.
conditions of temperature and cationic environment can be maintained during
the assay 
can be applied to high-throughput screening as its run isn’t time
of the flaw is in its methodology. An indirect competition between the
ligand and the probe when their binding sites are different leads to skewed
results. The TO displacement values would be an underestimate of that
ligand’s affinity 
characteristics of the studied molecule should not overlap with the
absorption or the emission spectra of the probe 
in telomere DNA can adopt numerous conformations which alters the binding
sites and hence a ligand’s affinity in vivo 
TRE (Telomere Repeat Elongation)
Repeat Elongation utilises SPR and quantifies the elongation of an immobilised
primer on the SPR sensor.
of a TRE assay
sequence used: 5’-biotin-AATCCGTCGAGCAGAGTTAG(GGTTAG)4
(10 mM HEPES, pH 7.4, 10mM MgCl2, 10mM NaCl, 2.5mM dNTP, 10mM
The telomeric sequence was immobilised onto a Biacore SA sensor chip. Cell
extracts that were telomerase positive were diluted in dNTP buffer and injected
over the SENSOR at 5μL/min at 37°C for periods of 1, 5, 10 and 30 minutes
to extend the telomeric oligonucleotide. The protein was then removed from the
sensor by injection of 1% SDS in HEPES buffer. In order to quantify the
extension of the oligonucleotide the baseline level was compared to that prior
to the injection of cell extract. A measure termed the elongation factor was
defined as the interval between the two aforementioned baseline responses. The
elongation factor increased linearly with incubation time.[Redman]
theory the TRE assay could be used to quantify inhibition of telomerase activity
G-Quadruplex stabilising ligands. Additionally, as it does not utilise PCR
amplification the TRE assay does not suffer from variations in efficiency
arising from the binding of ligands to duplex DNA.
mentioned herein involved a variety of analytical techniques that can be used to
study G-Quadruplex ligand binding. Calorimetric techniques provide a
thermodynamic profile of ligand:complex interactions from which various
stability data can be obtained.
ESI-MS can be used to quantify the relative abundances of ligand, G-Quadruplex
and ligand:Quadruplex in an interaction and clearly depicts preferential
interference and inhibition of Telomerase activity by potential G-Quadruplex
ligands can be studied by PCR stop and TRAP assays. The TRAP assay whilst
extremely useful suffers from reduced efficency that the PCR amplification
component introduces as ligands may bind to duplex DNA produced by PCR. TRE is a
new assay which utilises SPR and does not require PCR amplification.
dichroism provides information on the effects of ligand interaction on the
conformation of G-Quadruplex structures. On the addition of different ligands,
the alteration in response (molar ellipticity) for a corresponding wavelength
can be used to deduce these effect and characterise them as specific alterations
in specific regions of the Spectra. From this information the effects of various
ligands on the stabilisation and destabilisation of the G-Quadruplex can be
and G4-FID assays both use fluorescence spectroscopy in their working. They
involve attachment of fluorescent probes to the G-quadruplex forming
oligonucleotide. G4-FID measures the decrease in fluorescence energy and the %
displacement of the fluorescent dye whereas; FRET measures the increase in
fluorescence on increasing temperature.
assays are tailored here to evaluate the binding properties of ligand molecules
G4-DNA. Although these techniques only give semi-quantitative data (not giving
real parametric values e.g. affinity constants), the information provided in
invaluable to researches finding a potential target to stabilise the G-quadruplex
DNA and help them chose the best ligands for further analysis. Both methods are
easy to carry out and can be adapted to high-throughout screening of ligands.
These screenings test several molecule libraries at the same time. G4-FID does
not require specialised equipments; however, FRET may require several protocols
to use the PCR apparatus to measure melting temperatures. FRET is a more
commonly used technique when accessing G4-DNA ligand binding than G4-FID.
of assay will depend on a number of factors such as the ligands to be tested,
G-quadruplex structure (i.e. inter- vs intramolecular), quantity of sample,
ligand-quadruplex stoichiometry and the type of binding information sought.
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