Presented

By

Fatma M.Abdelkader Barakat

Pharmacist and Grad. Student
NCRRT
Atomic Energy Authority

AGENDA :
1-

Nanotechnology : An Introduction .

2-

NanoFlares Technology : An Overview .

3-

Aim of the work .

4-

Significance of this research work .

5-

Materials & Methods.

6-

Results & Discussion.

7-

Conclusion.

8-Summary.
9-

Current Research in the field .

NANOTECHNOLOGY

1- Nanotechnology & Nanomedicine

An Introduction :

Nanotechnology : Simply defined as the study and use

of structures between 1 nm and 100 nm in size.

This next definition from the National Nanotechnology

Initiative adds the fact that nanotechnology involves
certain activities, such as measuring and manipulating
nanoscale matter.

Nanotechnology is the understanding and control of

matter at dimensions between approximately 1 and 100
nanometers, where unique phenomena enable novel
applications.
Encompassing nanoscale science, engineering, and
technology, nanotechnology involves imaging, measuring,
modeling, and manipulating matter at this length scale.

An Introduction :

CONTD

Nanomedicine : the medical application

ofnanotechnology .
It

may be defined as the monitoring, repair,

construction and control of human biological
systems at the molecular level, using engineered
nanodevices and nanostructures.
Robert A. Freitas Jr, Nanomedicine ,
vol. 1 , 1999

Nanomedicine

ranges from the medical

applications of nanomaterials , tonanoelectronic
biosensors, drug delivery systems and possible
future applications ofmolecular
nanotechnology .

An Introduction : contd
Bionanotechnology refers to the study of how

biological "machines" work and adapting these

biological motifs to advance the goals of

nanotechnology .
For

example, DNA nanotechnology would be classified

as bionanotechnology because it involves working with

biomolecules on the nanoscale.

Nanobiotechnology, on the other hand, refers to the

ways that nanotechnology is used to create devices to
study biological systems.

For

example, medical technologies

involvingnanoparticlesas delivery systems or as

sensors are examples of nanobiotechnology since they
involve using nanotechnology to advance the goals of

In other words,
Nanobiotechnology is
essentially
miniaturizedbiotechnology

whereas

Bionanotechnology is a
specific application of
nanotechnology.

NanoFlare Technology : An Overview

1- What are Nanoflares ? ( Defenition )
2-

How do they target cells of interest ?

3-

What makes nanoflares unique ?

(Features of Nanoflares)
4-

How are Nanoflare probes being designed

?!
5-

How do Nanoflare (Smart flare ) probes

work?

NanoFlare Technology : An Overview

Over

the past decade, researchers have

investigated the conjugation of biomolecules to
inorganic nanomaterials which has led to the
development of hybrid materials with new
activities.

One

important class of hybrid nanomaterial is

so-called nano-flare system which is
composed of a Au NP functionalized with a
dense monolayer of oligonucleotides.

These

systems are capable of detecting specific

intracellular targets such as mRNAs and small
molecules in real time.

Nano flares : Tiny flares from the

Sun or from the Gold ?!

What are Nano-flares?

Answer 1
A nano-flare refers to a very smallsolar
flarethat happens in thecorona, the
externalatmosphereof theSun .

Answer 2
Nano-flares are a new class of intracellular
molecular probes.
Here we are only concerned with nano-flares that
are used as molecular probes. Hence we will only
deal with answer 2.

Defenition of a Nanoflare :
NanoFlare

is a spherical nucleic acid (SNA)

nanoconstruct capable of detecting
intracellular mRNA levels in live cells at the
single-cell level .

The

NanoFlare consists of a spherical gold

nanoparticle densely functionalized with
a monolayer of single-stranded DNA
(ssDNA) containing a 3 thiol that is
complementary to mRNA for a target gene.

The

ssDNA recognition sequence is

prehybridized to a shorter DNA complement
containing a fluorescent reporter
(reporter flare) whose fluorescence is
quenched based on its proximity to the gold
nanoparticle.
When target mRNA binds the recognition
sequence, the reporter flare strand is
displaced, providing a fluorescent readout.

Illustration of a Nano-flare probe

Watch the animation below to learn more

about these unique nanomaterials :

This animation describes the structure, properties,

and uses of the spherical nucleic acid (SNA), a
construct composed of a nanoparticle core and
densely packed highly oriented nucleic acid shell.
This structure has unique architectural-dependent
properties that set it apart from all other forms of
nucleic acids.
This includes the ability to cooperatively hybridize with
complementary nucleic acids and form stronger duplexes
than the same sequence of linear DNAand the ability to
efficiently transfect cell membranes without the need for
co-carriers.
These properties make SNAs extremely useful in :
1- In vitro medical diagnostics.
2- Intracellular detection.
3- Therapeutics.
N.B. Spherical Nucleic Acids (SNAs) were developed in the laboratory of

How do they target cells of interest ?

On

their surface are DNA sequences that

target messenger RNAs in cells.
When bound to their target, they release a
fluorescent signal.
Its the release of the signal flare that leads
to the name.
If the flare lights up, the target is present.
The

cells that are lighting up are those of

interest.

What makes nanoflares unique ?

Features of Nanoflares :
These

nanobioconjugates combine the discrete plasmon

resonances of AuNPs with the synthetically controllable and
highly selective recognition properties of DNA.

This

provide exceptional benefits for studying and

detecting disease in living systems, including:

1-

Cellular Uptake

2-

Stability :Nano-Flares are highly stable in a cellular

:Nano-Flares enter cells easily and lack

cytotoxicity since they exhibit signifcantly lower innate immune
response than comparable DNA systems delivered using commercial
transfection agents.

environment,with greatly increased resistance towards degradation by

nucleases .

These probes remain stable for long periods of time and have a low
background fuorescence or "false positive" signal (nano-flares exhibit high

signaling, are sensitive to changes in the number of RNA transcripts

present in cells )

Features of Nanoflares : contd

3- Specificity : they exhibit high target specifcity
and only giving off a bright signal when they encounter
their gene target .

4-Tailorabilty : Nano-Flares can be encoded to detect

any nucleic acid sequence of interest.

5-Real-time Monitoring : Nano-Flares function in

living cells and can thus detect specific gene level
changes in real-time, allowing a quantitative look at
changes occurring in live cells.

6- Efficacy :They are effective for single-gene and

multigene detection from a single nanoconstruct.

These unique properties thus

overcome many of the challenges to
creating sensitive and effective
intracellular probes which makes
Nanoflare systems highly valuable
for biological sensing and for the
development of tools for studying
many life-threatening diseases,
.including breast & prostate cancer

How are Nanoflare probes being

designed ?!
Synthesis of SNAAu NP conjugates.

The probes were designed using 13 nm gold nanoparticles that

can be densely functionalized with thiolated oligonucleotides or
oligonucleotide mimics as follows :

Citrate-stabilized particles are incubated with alkyl thiolfunctionalized oligonucleotides in water to form a low-density
monolayer.
By incubating the nanoparticles in aqueous solutions with
successively higher concentrations of salt (typically 0.151.0 M) and
surfactants over 12 h, a high-density SNA shell is formed.

Design of Nanoflare probes :

Nanoflares were designed using 13 nm Au NP
since this size particle is :
An efficient quencher.
can be densely functionalized with
oligonucleotides.
Does not efficiently scatter visible light.
which is important for designing optical
probes with
minimal interference.

Schematic of NanoFlare structure and function.

The NanoFlare contains a monolayer of antisense DNA
(recognition sequence) adsorbed to the surface of a 13-nm
spherical gold nanoparticle.
A reporter flare sequence is hybridized to the recognition
sequence, which contains a fluorophore (red).
The dye is quenched in close proximity to the gold surface.
The reporter flare is displaced when complementary mRNA
(blue) binds the recognition sequence, providing a fluorescent
signal.

Smart Flare RNA Detection Probe

How do Nanoflare probes work ?

The probes, oligonucleotides specific to a target RNA that are
bound to AuNPs , are incubated with cells overnight.
Cells endocytose the nanoconjugates, which bind to their
complementary RNA target, releasing a fluorophore that can be
detected via flow cytometry, microscopy, or any downstream
method that relies on fluorescence.
The cells exocytose the particles after a few days and thus
recover completely from treatment.

These new RNA detection probes can be used to visualize

RNA expression in live cells at the single-cell level .

SmartFlare RNA
Detection Probes

New SmartFlare RNA Detection

Probes from EMD Millipore offer an
alternative option for cell sorting by
allowing fluorescent detection of
internal RNA species without harm
to the cell no antibodies required

Named as one of
Top Innovation of
2013
by The Scientist
Magazine

Smart flare video

How to use the smartflare probe ?

Just Mix, Incubate, Read and Reuse.
Sample prep is nonexistent; simply add the SmartFlare Probes
to your cultures, incubate overnight and detect the next day.
Over time, the probes will exit the cell, without any obvious
adverse effects, allowing for subsequent downstream assays.
SmartFlare RNA Detection Probes make your work
effortless:
No sample prep
No nucleic acid amplification
No transfection reagents
No cell lysis
No toxicity

Terms to be familiar with :

Smart flare RNA detection probe = Nanoflare =SNA
=Spherical Nucleic Acid = Nanoconjugate = Gold
nanobeacons = Gold nanoparticle-based molecular beacons
= Oligonucleotides functionalized AuNPs

Theranostics:

A term made up by combining the two

terms therapeuticsanddiagnostics which is a proposed
process of diagnostic therapy for individual patients that
involves testing them for possible reaction to a new
medication and to tailor a treatment for them based on the
test results.

LNA : Locked nucleic acid ,

inaccessible RNA is a

modified RNA
nucleotide in which the ribose ring is constrained by a
methylene linkage (extra bridge) between the 2-oxygen
and the 4-carbon .

The locked ribose conformation enhances base stacking and

backbone pre-organization
significantly increases the
hybridization properties of oligonucleotides (
melting
temperature ) & also increases binding affinity for
complementarity sequences .

Benefits of using LNA include :

1- ideal for the detection of short RNA & DNA targets.
2- increases the thermal stability of duplexes.
3- Capable of single nucleotide discrimination.
4- Resistant to exo- and endonucleases resulting in high
stability in vivo and in vitro applications .

Aim of the work :

Characterization

of the target binding ability and the binding rate

of nanoflares using survivin, a well studied gene associated with
the diagnosis and treatment of cancer, as a target.

The

specificity of the nanoconjugates for their complementary

target in an extracellular environment will be studied.

Investigation

of the intracellular interactions of nanoconjugate with

endogenous mRNA elucidating its mechanism of gene regulation.

Herein,

authors demonstrate, for the first time, a single material

capable of both regulating and detecting mRNA.

Significance of this research work :

This

work is important to the study of biologically active

nanomaterials such as the nano-flare and is a first step
towards the development of an mRNA responsive
theranostic material.

Additionally,

it also demonstrates that gold

nanoparticles conjugated to fluorophore-labeled
oligonucleotide duplexes readily enter cells, and
function as an antisense nanoconjugates which
provide a fluorescence signal that correlated with
the presence and abundance of a specific
intercellular nucleic acid and outperforms the
uptake ability , stability and gene silencing ability
of molecular beacons (antisense oligonucleotides
probes) .

Results and Discussion

Nanoconjugates were designed with several features that make

them well suited for both mRNA regulation and detection (Figure 1)

The antisense oligonucleotide (recognition sequence )portion of the

nanoconjugate targets an mRNA region selected to control
expression of survivin gene .

Additionally, the nanoconjugate recognition sequence is a DNALNA chimera, where the LNA bases serve to increase mRNA
binding affinity , thereby increasing detection and regulation
efficiency of the probes.

Finally, the nanoconjugate contains fluorophore-labeled flare

oligonucleotides (reporter flare ) that are designed to dissociate
upon target binding.

The bound flare is quenched by the AuNP, so the dissociation can

be
detected as an increase in the fluorescence intensity .

(Figure 1 ) mRNA depletion and detection using survivn nanoconjugates .

orange

13 nm AuNP

DNA

Black

LNA

green

mRNA

purple

target mRNA

Cy5

Red

purple or pink

when quenched or
released,
respectively.

Results and Discussion Preparation of

nanoconjugates

To prepare the nanoconjugates, citrate-capped gold

nanoparticles were functionalized with thiol-terminated
antisense oligonucleotides .

A high surface coverage (90 10 oligonucleotides per

NP) was achieved by slowly increasing the Na Cl conc. to
0.3 M .

These nanoconjugates were purified by centrifugation,

and a short, complementary, Cy5-labeled oligonucleotide
was added (Figure 1).

A second nanoconjugate (86 4 oligonucleotides per

nanoparticle)
that lacks the full antisense sequence was also
synthesized to serve as a control.

Examining the response of the nanoconjugates (survivin

& control )
to synthetic DNA targets (Figure 2 )

Figure 2 (contd)

The nanoconjugate (1nM) was

added to a solution of
phosphate buffered saline (pH
7.4) and , fluorescence of the
solution was measured .

In the absence of a target, both

solutions of nanoconjugates
exhibited a low fluorescence
signal.

The addition of the survivin

target results in a 3.7 fold
increase in the fluorescence
associated with the survivin
nanoconjugate .

In the case of the control

nanoconjugate, little response

The sequence specific

increase in fluorescence
is consistent with flare
release from the gold
nanoparticle surface.

Testing the nano-flare design using synthetic

complementary
targets demonstrates that the probes respond with
a 3.7 fold increase in fluorescence signal upon
target recognition and binding ( Fig. 2)
In contrast, the signal does not change in the
presence of a
Noncomplementary target and is of comparable
magnitude to
background fluorescence.
These results thus demonstrate that nano-flares
are efficient at signaling the presence of a specific
target .

Characterization of the target binding rate and

ability of the nanoconjugates :

In order to detect target

mRNA it is critical for the
nanoconjugates to respond
to their target rapidly.

In order to investigate the

rate of flare release they
added an excess of target to
a solution containing the
nanoconjugates and
monitored the change in
fluorescence over time.

Fluorescence
increases rapidly,
reaching completion
in 10 minutes (Fig.2)

Fig.3 : Experiments to characterize the

specificity of the nanoconjugates for their
complementary target

Nanoconjugates of an analogous
design but containing a different
oligonucleotide sequence (see Methods
Section) were challenged with a series
of targets containing four, three, two
or one base pair mismatches.

Following incubation with these

mismatch oligonucleotides,
fluorescence was measured to
examine nanoconjugate binding and
subsequent flare release.

Nanoconjugates that were treated

with targets that contain four, three,

or two mismatches show little

increase in fluorescence signal,
similar to nanoconjugates that were
treated with buffer.

 Nanoconjugates that were

treated with targets

containing one mismatch
show a fluorescence
response .
However, this signal is
easily distinguished from
that observed with the fully
complementary target
because it is 48% less
intense.
These results demonstrate

that the nano-flares can be

used to distinguish targets

Investigation of interacellular interaction of

nanoconjugates with endogenous m RNA

After the specificity and selectivity of the nanoconjugates

in an extracellular environment were confirmed,
researchers next studied

theirThe
activity inside living cells as follows :
HeLa cells were harvested and washed, and the cell-associated fluoresc
HeLa
The cells were harvested and washed, and the cell-associated fluoresc

HeLa cells treated with survivin nanoconjugates are 1.7 0.1

times more fluorescent than cells treated with control
nanoconjugates (Fig 4).

Fig. 4

Survivin nanoconjugates respond to

intracellular mRNA.

Top

Confocal fluorescence
microscopy of HeLa cells
treated with either survivin or
control nanoconjugates

Cy5 fluorescence associated with flare

(left, in red) and bright field Cy5
fluorescence overlay (right) are shown .

Bottom

Flow cytometry data for HeLa

and C166 cells treated with
both nanoconjugates.

The fluorescence was

normalized to the cell
population treated with
control nanoparticles.

The experiments were repeated

with C166 cells (a mouse cell
line lacking a human survivin),
both survivin and control
nanoconjugates exhibited similar
cell associated fluorescence.

Taken together, this

series of experiments
shows that the
nanoconjugates can be
used to
distinguish different cell
populations based on
mRNA levels ( gene
expression profiling )

Figure 5 :Investigation of the ability of

nanoconjugates to regulate intracellular mRNA levels

N.B. Survivin levels were normalized to actin mRNA, an off-target

gene

Figure 5 : The effect of nanoconjugates on survivin mRNA

levels in HeLa cells.

N.B. The relative abundance of survivin mRNA was determined by

RT-PCR and normalized to expression levels in untreated cells .

Survivin mRNA levels were not significantly changed

when cells
were treated with either a low conc. of survivin
nanoconjugates
(0.5 nM) or any conc. of control nanoconjugates.

These
data are
with agene
doseand sequenceWhen
compared
to consistent
mRNA detection,
regulation
appears
to require
dependent
reduction of mRNA.

relatively high concentrations of nanoconjugate (5 rather

than 0.5 nM) & relatively long exposure times (4 days
rather than 6 hours).

This is likely due to several factors including:

Cellular feedback loops that compensate for mRNA loss.
Efficient detection of mRNA when only a small percentage
of the
total mRNA population is bound.

The direct observation of reduced mRNA levels (Fig. 5)

can elucidate the mechanism of nanoconjugate gene
regulation.

In previously studied molecular antisense, mRNA binding

recruits
( RNase H) which degrades the DNA/RNA hybrids which
makes the
mechanism of gene silencing catalytic .

In some other research work using similar

nanoconjugates, researchers observed depletion in
protein levels, which can be caused by either translation
Here, they
found depletion.
that nanoconjugates
inhibition
or mRNA
cause mRNA depletion , a phenomenon
that has been observed with traditional
antisense oligonucleotides.

Methods

Methods :
1- Oligonucleotide synthesis:

Oligonucleotides were synthesized with an Expedite

8909 Nucleotide Synthesis System (ABI) using standard
solid-phase phosphoramidite methodology.

All oligonucleotides were purified by reverse-phase high

performance liquid chromatography (HPLC).

The oligonucleotide sequences used in this study are

LNA, some others are DNA.

N.B. These nucleotides sequences including the sequences of :

(Target DNA, Control , Survivin , Flare, Full complement , Single mismatch , two
mismatch , three mismatch, four mismatch ) are listed in the article .

( please refer to methods section )

Methods :Nano-flare probes preparation :

2- Oligonucleotide gold nanoparticle conjugates

(functionalization process )

3- Oligonucleotide loading :

The concentrations of the the purified oligonucleotide

functionalized AuNPs ( nanoconjugates ) were
determined by UV/Vis spectrophotometry by measuring
at ( = 524 nm)

The nanoconjugates were then treated with KCN

solution (0.1 M) to oxidatively dissolve the gold particles
and liberate the surface coordinated oligonucleotides.

Oligonucleotide concentration was determined using a

commercially available kit .

The average no. of oligonucleotides conjugated to a

nanoparticle was calculated = conc.of
oligonucleotides / conc.of nanoparticles.

4- Flare duplex formation

5- Fluorescence spectroscopy

Fluorescence measurements were recorded on a Jobin

Yvon Fluorolog FL3-22 exciting at 633 nm and
measuring emission from 650 to 750 nm in 1 nm
increments .

Static fluorescence was measured on samples

containing nanoconjugates (1 nM) in PBS with 0.1 %
Tween-20.

The complementary target (200 nM) was added and the

solutions were remeasured.

The time course experiment was carried out by treating

solutions of nanoconjugates with target and measuring
fluorescence (670 nm)
in 20-second increments for 1 hour .

6- Cell culture

HeLa (human cervical cancer) and C166 (mouse

endothelial) cells were obtained from the American Tissue
Culture Collection
(ATCC) and were grown in Essential Minimum Eagles
Medium
(EMEM) and Dulbeccos modified Eagles medium (DMEM),
respectively

Both media contained 10 % heat inactivated fetal bovine

serum, and the cells were maintained at 37C in 5% CO2 .

Cells were seeded 1 day prior to treatment with the

nanoconjugates.

7- Flow cytometry

The next day, cells were

To normalize for cell-type, the values were reported as a

fraction of
the control nanoconjugate-treated cells.
The data reported in ( Figure 4) represents the average cell
associated fluorescence for a population of cells .

8- Imaging

9- RT-PCR

RT-PCR (contd)

The relative abundance of each mRNA transcript was

normalized to actin expression and compared to
untreated cells to determine the
increased expression .
The standard deviation for this data was calculated from
3 independent experiments.
The primers used to make cDNA in this experiment
were:
Survivin forward, 5 -ATG GGT GCC CCG ACG TTG - 3
Survivin reverse, 5 - AGA GGC CTC AAT CCA TGG - 3
Actin forward, 5 - ATC ATT GCT CCA CCA GAA CG - 3
Actin reverse, 5 - AAG GTAGAT AGA GAA GCC AAG - 3.

Authors Conclusions :

In this research paper authors have designed,

synthesized, and characterized a nanoconjugate that can
both detect and regulate intracellular mRNA levels .

These nanoconjugates signal the presence of target

mRNA with the release of a flare sequence, which results
in an increase in fluorescence.

The target binding and response of the nano-flare is rapid

and capable of recognizing single base-pair mismatches.

The same nanoconjugates deplete mRNA levels by as

much as 92% 4 in a dose- and sequence-dependent
manner, consistent with enzymatic degradation of
targeted mRNA.

Authors Conclusions (contd)

Although similar gene regulation and detection

strategies have been used in the past , this work
represents the first combination of gene regulation and
detection in a single material.

Moreover, the experiments show for the first time that

oligonucleotide functionalized particles directly deplete
mRNA levels which indicates that this mechanism is
important, not only for traditional antisense
oligonucleotides, but also for nanoconjugates that
function in gene regulation pathways.

These nanomaterials are a promising first step towards

the development of mRNA directed theranostics and
are expected to combine the advantages of gene
therapy with personalized medicine.

In summary, we have described a new class of

intracellular probe termed Nano-flares.
Nano-flares are novel and potentially very useful since
they are the only probe that combines cellular
transfection, enzymatic protection, and detection of
mRNA in living cells and quantification.
Nano-flares will also be of utility in other areas such as
cell sorting, gene profiling, and real-time drug
validation studies.

Finally,

this study also demonstrates the important

and often surprising role nanostructure plays in
controlling a materials biological function and supports
the continued development of nano-flares as probes to
increase our knowledge in understanding of
fundamental cellular events in biology and diseases.

References

Amelie Heuer-Jungemann, a. P. K. H., a Tom Brownbc and a. A. G. Kanaras (2013). "Gold

nanoparticles and fluorescently-labelled DNA as a platform for biological sensing , "
Nanoscale .

Joshua I. Cutler , E. A., and Chad A. Mirkin (2012). "Spherical Nucleic Acids." Journal of
American Chemical Society :134(3): 13761391.

Prigodich AE, S. D., Massich MD, Giljohann DA, Lane BC, Mirkin CA (2009). "Nanoflares for mRNA regulation and detection ACS Nano : 3(8): 2147-52.

Seferos DS, G. D., Hill HD, Prigodich AE, Mirkin CA (2007 )

"Nano-flares: probes for transfection and mRNA detection in living cells."

J Am Chem Soc. 129((50)): 15477-9.

Tiffany L. Halo, K. M. M., Nicholas L. Angelonic, Yilin Xuc, Wei Wangc, Alyssa B.
Chinena, and E. S. Dmitry Malinh, Vincent L. Crynsh, Chonghui Chengc, Chad A. Mirkin
and C. Shad Thaxton (2014). "NanoFlares for the detection, isolation, and culture of live
tumor cells from human blood PNAS : 111(48): 1710417109.

Zheng D, S. D., Giljohann DA, Patel PC, Mirkin CA (2009 ). "Aptamer nano-flares for
molecular detection in living cells." Nano Lett 9(9): 3258-61.