APTAMERS

BY-
SURABHI VERMA (16/PBT/007)
SURYAKANT TIWARI
(16/PBT/008)
SWATI SINGH (16/PBT/009)
 What is an APTAMER?

short single stranded oligonucleotide

or peptide molecules
can bind to pre-selected targets
including proteins and peptides with
high affinity and specificity
can assume a variety of shapes
used as sensors and therapeutic
tools
APTAMERS CAN BE CLASSIFIED AS-
 AptaBiD

technology for biomarkerdiscovery

based on multi-round generation of
an aptamer
involves three major stages:

differential multi- mass

aptamer-based
round selection of spectrometryide
isolation of
aptamers for ntification of
biomarkers from
biomarker of target biomarkers
target cells
cells
 it produces synthetic affinity probes
(aptamers) simultaneously with
biomarker discovery
aptamers are developed for cell surface
biomarkers in their native state and
conformation
aptamers can be directly used for cell
isolation, cell visualization, and tracking
cellsin vivo
 HOW DO APTAMERS WORK?
types of aptamers have very similar properties but are
distinctly unique.

selected against small molecules, toxins,

peptides, proteins, viruses, bacteria, and even whole cells.

bind withhigh specificity and affinity- once they have

determined their target, they then bind to it with a strong
bond.

Because theystructurally conformto bind to their targets,

this gives aptamers awider range of possible targetswhen
compared with antibodies, whichrequire antigens and
epitopesalong with animmune responsefor their targets.
 DNA &RNA APTAMERS
most common type of aptamers
chosen from a pool of random nucleic acid
sequences and put through several cycles
for optimization in aprocess called SELEX
single stranded and typically around15-60
nucleotide basesin length & longest
sequences have been selected at220
nucleotides
bond by complementaryRNA base
pairing
 secondary structures such as short
helical arms and single stranded loops
due tobase pairing
tertiary structures allow aptamers to bind to
targets viavan der Waals forces, hydrogen
bonding and electrostatic interaction
aptamer-target complexes - entire aptamer
folds into astable complex with the target
ligand
3D structure allows aptamers tofunction like
antibodies
Aptamer to protein binding ability is in
the range of monoclonal antibodies
 DNA aptamers are inherentlymore
stable, cheaper, and easier to produce
RNA also requiresreverse transcription,
in which RNA must be converted base-for-
base into DNA - whereas DNA does not
require this extra stage in the SELEX
process
DNA and RNA aptamerscan also differ
in sequence and folding pattern even
when selected for the same target
DNA and RNA aptamers bindusing their
entire sequence
DNA Aptamer RNA
Aptamer
 " Most people think of nucleic
acids as information holders, not
globular things like proteins...what
our work proved is that when you
do SELEX...you get molecules that,
even though they're chemically
made out of nucleic acid, are more
like proteins than nucleic acids "

- Dr.
Larry Gold
 PEPTIDE APTAMERS
small, simple peptides with a single
variable loop regiontied to a protein on
both ends
bind to their targets with this variable loop
region
being tied to loop region reduces the
flexibility of peptide aptamers and
effectiveness
newest form of aptamers,much is still
unknownabout these structures
SCREENING METHOD FOR
APTAMERS: SELEX
 APTAMER MODOFICATION

increase aptamer half-lives or for

enhanced therapeutic abilities
major problem - their small size
results in susceptibility to renal
filtration and nuclease degradation ,
cause very short half-lives if left
unmodified
 Nuclease degradation - Aptamers, as nucleic-acid
based molecules, are susceptible to enzymes
callednucleases that cleave nucleic acid bondsand
can be found throughout the body. This destroys the
nucleic acid structure. Aptamer structures can
bechemically modified, with modifications to the
nucleotide sugars and internucleotide linkages
increasing nuclease resistance. "Capping" at the ends
of the chains that involve reversing polarity is
another strategy, along with inverting nucleotides.
Modified nucleotides with functional groups such as
fluoro, amino, orO-methyl groups also increase
nuclease resistance.
 Renal Filtration -Because of their small
size, aptamers are subject tofiltration by
the kidneys. Most aptamers are5-15 kDa
long, or around 15-50 nucleotides.
Aptamers forcommercialapplications are
often shortened as much as possible to
reduce manufacturing costs. The cutoff for
renal filtration is around30-50 kDa, so
conjugation to a polymer with high molecular
mass calledpolyethylene glycol (PEG) can
make aptamers resistant to renal
filtration.
 Conjugation - Aptamers can be conjugated,
orjoined with, additional units that result in
new properties for the aptamer. For example,
a non-nucleic acid unit can be joined to an
aptamer to allow for additional bondsto be
formed between the target and the aptamer
that would not be possible without the
additional unit.
Additionally, aptamers can be conjugated to
drugs, functioning asdrug-delivery systems
Other examples of this concept include
conjugation to toxins and small interfering
RNAs (siRNAs)
RECENT ADVANCES IN SCREENING
METHODS OF APTAMERS
1. CE Microfluidic Chips
2.Sol-Gel Microfluidic Chips for
Screening of Aptamers
3.Magnetic-Bead-Based Microfluidic
Chips for Screening of Aptamers
Advantages of Aptamers
Can disrupt protein- protein interactions.
Aptamers therapeutics which are used are given
by the subcutaneous route.
They are stable at room temperature so their
storage at room temperature is possible.
Aptamers have wide therapeutic margins, more
stable, have modulated pharmaco-kinetic activity.
Very low toxicity and low immunogenic activity.
Can produce chemically and in readily scalable
process.
Small sized aptamers can easily and efficiently
get entered into the biological compartments.
Aptamers reversibly get denatured and
 Disadvantages of aptamers
Pharmacokinetic properties and other
systemic properties are very hard to
determine and are variable.
They have small size, so they can easily
pass from the renal filtration, thus they
have a very short half-life. Some un-
modified aptamers are also highly
susceptible to serum degradation. This
technology is now covered by a single
intellectual property portfolio.
 DIFFERENCE BETWEEN
APTAMER AND ANTIBODY
Aptamers are capable of greater specificity and affinity than
antibodies
They can easily be modified chemically to yield improved,
custom tailored properties. For instance, a reporter and
functional groups and PEG can easily be attached to the
aptamer in a deterministic way. In fact, they can even be
combined with antibodies
Similarly, their ADME properties can be readily tuned by
conjugation to other groups (PEG, etc.)
Their small size leads to a high number of moles of target
bound per gram, and they may have improved transport
properties allowing cell specific targeting and improved
tissue penetration
They are much more stable at ambient temperature than
antibodies yielding a much higher shelf life
They can tolerate transportation without any special
 LIMITATIONS IN APTAMER

Aptamer degradation
Aptamer excretion from the bloodstream by
renal filtration
Control of the duration of action
Interaction of aptamers with intracellular
targets
Generation of aptamers using unpurified
target proteins
Aptamer cross-reactivity
Automation of aptamer generation
 CURRENT STATUS OF APTAMERS IN DIAGNOSTICS AND THERAPHY

Mono- and polyclonal antibodies can sometimes

be successfully replaced by aptamers
Aptamers can recognize a membrane-
immobilized protein in Western blotting
protocols more effectively than antibodies
ELISA protocols are also more sensitive when
aptamers are used instead of antibodies
In contrast to antibodies, aptamers can be
selected against non-immunogenic and toxic
substances
 Aptamers are promising therapeutic
agents
Aptamers are also used as
recognizing elements in biosensors
Aptamer-based protocols of
treatment of viral diseases are under
development