CONCEPT OF APTAMER AND ITS APPLICTION

Presented by : ACHYUT BORA and NEELOTPAL SHARMA

APTAMER Aptamer (aptus: “to fit”& mer: “smallest unit

of repeating structure”). Single stranded folded oligonucleotides and

peptide that bind to molecular (protein) targets with high affinity and specificity.

They are similar to monoclonal antibodies. Provide opportunities for structure-based drug

design strategies relevant to therapeutic intervention.

Can be prepared using SELEX(Systematic Evolution Ligands by Exponential Enrichment ).

Based on their three-dimensional structures, Aptamers can well-fittingly bind to a wide variety of targets from single molecules to complex target mixtures or whole organisms.

They range in size from 20 to 80 bases(6-26 kDa).

APTAMER PRODUCTIONSELEX Systematic Evolution 0f Ligands by Exponential Enrichment This method, described primarily in 1990(Ellington, A.D & Szostak, J.W., 1990)

SELEX contd

Aptamer are produced by an in vitro selection or systematic evolution of ligand by exponential enrichment(SELEX).

It will produce ssRNA with specific binding target. This is an iterative process of binding, partitioning, amplifying novel nucleic

acids and regeneration.The first step is to synthesize a large pool of nucleic acid molecules made

from average of 15-40 bases of random sequences that are flanked by primers 5’ and 3’ end.

The pool of DNA is transcribed into an RNA pool and it subsequently exposed to the target ligand of interest.

Affinity column chromatography removes unbound sequences identify the strongest binding sequences and bound sequences are eluted and amplified by RT-PCR .

PROPERTIES OF APTAMERS RNA/DNA or Protein oligonucleotide molecules. Ability to bind to proteins with high affinity and specificity. Adopt wash number of 3-D shapes- which enables them to bind tightly to specific molecular

target. Usually very small in size but can easily distinguish between very closely related protein also. Highest specificity and affinity to the target. Small size and similarity to endogenous molecules. Ability to disrupt interaction between proteins, High specificity and high affinity to the target and are capable of penetrating tissues then

antibodies because of their smaller size. Low or non immunogenic and non toxic molecules. Production of aptamers is done in-vitro using automated synthesis. Rationally design duration of action. Less cost of oligonucleotide synthesis. Function at different temperature, Ph and buffers. Unlimited shelf life.

RNA APTAMER DNA APTAMER PEPTIDE APTAMER

FROM COMPLEX SECONDARY AND TERTIARY STRUCTURE

FROM COMPLEX SECONDARY AND TERTIARY STRUCTURE

STRUCTURE CONSTRAINS BY SCAFFOLED

FROM DIVERSE 3D STRUCTURE LESS DIVERSE 3D STRUCTURE THAN RNA APTAMER

3D STRUCTURE CONSTRAINTS BY SCAFFOLD

BIND TARGET WITH THE ENTIRE SEQUENCE

BIND TARGET WITH THE ENTIRE SEQUENCE

BIND TARGET VARIABLE REGION ONLY

BIOSENSEOR, DIAGONOSTIC, THERAPEUTICS APPLICATIONS

BIOSENSEOR, DIAGONOSTIC, THERAPEUTICS APPLICATIONS

BIOSENSEOR, DIAGONOSTIC, THERAPEUTICS APPLICATIONS

COMPARISON OF RNA, DNA AND PEPTIDE APTAMERS:

Aptamer Antibodies

Aptamer are oligonucleotide and protein Antibodies are protein in nature.

Uniform activity regardless of batch varies from batch to batch. Investigator determines target site of protein Immune system determines target site of

protein.Wide variety of chemical modifications to molecule for diverse functions

Limited modifications of molecule

No evidence of immunogenicity. Significant immunogenicity They are more stable at high temperature and they can be regenerated easily after denaturation.

Temperature sensitive

Entire selection is a chemical process carried out in vitro and can therefore target any protein .

Selection requires a biological system, therefore difficult to raise antibodies to toxins (not tolerated by animal) or non-immunogenic target.

Aptamers are single stranded DNA or RNA oligonucleotide or peptides.

Antibodies are monoclonal or polyclonal.

COMPARISION BETWEEN APTAMER AND ANTIBODY

APPLICATION OF APTAMER

In 2004, the approval by the Food and Drug Administration (FDA) of Macugen, a vascular endothelial growth factor (VEGF)-specific aptamer, for the treatment of neovascular (wet) age-related macular degeneration (AMD), is a prominent landmark in the application of aptamers.

This drug is a pegylated aptamer, a single strand of nucleic acid with specificity to VEGF165, which plays a critical role in angiogenesis and permeability.

Additionally, Regado Bioscience has developed REG1 as a new aptamer drug for anticoagulation, and this aptamer drug is currently in Phase II clinical trials. REG1 consists of two components: RB006 (coagulation factor IXa-specific aptamer) and RB007 (oligonucleotide antidote of the RB006 aptamer). RB006 is a 2′-ribo purine/2′-fluoro pyridimidine aptamer and is conjugated to a 40 kDa PEG to protect the aptamer against nuclease-mediated degradation.

Aptamers: as drug therapeutics

Due to specific and tight affinity to target molecules, and low or no immunogenicity and toxicity, aptamers are expected to be effective therapeutics reagents.

Aptamers that bind to internalized cell surface receptors have been exploited to deliver drugs and a variety of other cargo into cells.

For example:(a)The prostate-specific membrane antigen (PSMA) is an important prostate cancer marker. The dual aptamer probe—an A10 aptamer for PSMA(+) prostate cancer cells, and a DUP-1 aptamer for PSMA(−) prostate cancer cells—was invented, and a drug loaded dual aptamer complex was constructed by loading doxorubicin, an anticancer drug, onto the A10 aptamer strand. As a result, the doxorubicin can be effectively introduced into the prostate cancer cells. (b) Design of an siRNA-aptamer conjugate via a modular streptavidin bridge using an anti-PSMA aptamer for prostate cancer cells (LNCaP).

APTAMER: as DRUG DELIVERY SYSTEM

Aptamer: uses in Bio-Imaging

Another application is bio-imaging, using an aptamer that is conjugated to a fluorophore, or other materials such as gadolinium, which is useful for magnetic resonance imaging (MRI).

Using aptamers as imaging agents has the advantage of their being non-toxic, because oligonucleotide moieties are present in the human body.

As aptamers have high specificity for their target, accurate targeting, and rapid diffusion through the blood circulation, use of these molecules can increase the certainty of the results obtained during diagnosis or clinical analysis.

Based on these advantages, aptamers have been studied as imaging agents for cell imaging as well as single-protein imaging.

Aptamers: Ease in Western Blot Analysis

A Western blot analysis is an analytical technique routinely used to quantify specific proteins. The procedure includes complicated and elaborate steps and requires many reagents, such as two types of antibodies.

Hah’s group published a new aptamer-based Western blot strategy that has reduced the procedure to one step, and easily detects the target protein using only one aptamer.

Instead of two types of antibodies, the QD-conjugated RNA aptamer specific for the His-tag was employed. This method has the advantages of requiring less time, not requiring antibodies or 32P, and introducing the possibility of multiplexing detection.

(a) The conventional Western blot analysis, (b) The aptamer-based Western blot analysis.

APTAMERS AS DIAGNOSTIC TOOLS

As Aptamers are high affinity and specificity, small size, little immunogenicity, stable structures and ease of synthesis it can be used as diagnostic tools.

Aptamer based detection assays are expected to detect low concentration pathogens than conventional antibody based detection assay such as ELISA.

e.g

VIRUS TYPES OF APTAMER TARGET SPECIFIC MODE OF ACTION

HIV-1 Strain R5 2’-fluropyrimidine containing RNA aptamer

Glycoprotein 120 Inhibit cell to cell movement and virus to cell infection

Hepatitis B virus Peptide aptamer C1-1

Viral coat protein Inhibiting virus replication by blocking capsid formation.

ADVANTAGES

Produced chemically in a readily scalable process Not prone to viral or bacterial contamination Non-immunogenic more efficient entry into biological compartments Able to select for and against specific targets and to select against cell-surface

targets Can usually be reversibly denatured Dyes or functional groups can be readily introduced during synthesis

DISADVANTAGES

Pharmacokinetic and other systemic properties are variable and often hard to predict

Small size makes them susceptible to renal filtration have a shorter half-life Unmodified aptamers are highly susceptible to serum degradation Aptamer technologies are currently largely covered by a single intellectual

property portfolio

Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention

Recent advances in the chemical modifications of nucleic acids suggest that one of the major barriers to use, stability, can be overcome

The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and therapeutic application

We should expect more aptamers to be isolated in the near future against an ever increasing repertoire of targets, using these different SELEX approaches with increased speed and efficiency

Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades

CONCLUSION