Aptamer Hari pankaj vanam aptamer a plethora of opportunities

APTAMERS“A Plethora Of Opportunities”

HARI PANKAJ VANAM

Assistant Professor

Department of Microbiology

27th MARCH 2016

OBJECTIVES

What are Aptamers- definition, Properties.

History of Initial Aptamers & SELEX

mAbs Vs Aptamers (hype / humbug!)

Types of Aptamers.

SELEX Process and Its modifications.

Potential applications- Where are we ?

3/27/2016Hari Pankaj Vanam #Micro@ BMC

Latin-aptus (to fit), Greek meros - part

Short ssDNA, RNA - oligonucleotides (15-75) or peptides

Unambiguous binding affinity they discriminate for their

specific targets with high precision!!high specificity .

(unsuitable for Ab).

Essentially as structural /Molecular recognition.

Kd of aptamer-protein binding: 1pico molar to 1 nanomolar.

Chemical Abs untouched areas of therapeutics.

Unlimited spectrum; unbound by limitations of immunogenicity.

What are Aptamers?

Ex: of targets: small

molecules, Ions, toxins,

peptides, proteins,

viruses, bacteria, and

even whole cells.3/27/2016Hari Pankaj Vanam #Micro@ BMC

Aptamers doesn't bind to targets by canonical base-pairing like oligonucleotides used in PCR,

because it's target is NOT DNA.

They often base pair internally, making stems and loops that position bases in optimal locations.

Generated chemically-Controllable and modified. SELEX!!!


APTAMERS ARE POLYANIONS

They like to stick to polycations (histones, DNA

BP, Heparin BPs etc.,)

By including -polyanion (dextran sulfate)in

SELEX can increase specificity by competing for

nonspecific binding.

Including non-natural bases in DNA helps

increase sensitivity. Modified bases!!!3/27/2016Hari Pankaj Vanam #Micro@ BMC

A History of Aptamers

Concept of “joining NA with proteins”: 1980s-Studies from

HIV & Adeno ”RNAs are encoded by these viruses to

bind to cellular proteins ”with high affinity and specificity

–hence known as aptamer ( coined by Ellington).

1900 SELEX -Systematic Evolution of Ligands by

Exponential enrichment (SELEX) process, -streamline

aptamer-isolation process-improve efficiency/optimization.

past two decades over 25+ different SELEX processes -

All follows GOLD & TUERK’s (1993 patent) basic

Approach.

Currently, over 4,000 published articles PUBMED-

The New Beginning !!!


Genesis of SELEX –

Prof .Gold L & Student Tuerk

Intitial aptamers -SELEX:

RNA ligands to BØ T4 DNA pol.

Science 1990; 249:505-10, Patent in 1993.

Awards-European Inventors of the Year 2006.

Experiment: A randomized pool of ~65,500 N

sequences underwent the process, and two of these

sequences were isolated.

Results led to the concept that “NAs could be used

to recognize proteins.”

Ellington Andy, Szostak JW. In vitro selection of RNA

molecules -that bind specific ligands, Nature 1990 .

Gerald Joyce –California independently- developed

SELEX.

Scostak

Gerald

Joyce

Ellington Andy

Univ. of Colorado


Folded DNA becomes Trojan

horse to attack cancer


http://www.newscientist.com/article/mg21528785.400-folded-dna-becomes-trojan-horse-to-attack-cancer.html

Timeline of mAbs Vs Aptamers

R.Yallow

RIA-for

insulin

Kohler &

Milstein

mAb’s

Yalow

Nobel

Kohler &

Milstein

NobelIHC common surg

pathology tech

Aptamers

invented

Tuerk & Gold

Patent-SELEX

Several

aptamers

and

clinical

app’ns

tested

SELEX

Automation

towards

efficient

process

=Less time

Macugen

and other

products

Somagen

etc.,


Property mAb’s Aptamers

1.Stability: -limited shelf life.

-easily denatured.

-require

refrigeration

-Indefinite shelf life.

Withstand denaturation/

renaturation

-temperature resistant

stable @ room temp !!

2.Nuclease

Degradation

A.A’s not affected are subjected to nuclease

degradation limits half-life.

(ways to modify)

3.In vitro

capability

use of a live animal

-limiting

adaptability.

IEC/CPCSEA etc.,

In Vitro SELEX.

Manipulated to adapt

to virtually any set of

conditions.

4. Immuno-

genicity

Foreign: evoking an

undesired immune

response.

Repeated doses?

DNA and NA are native: Do

not evoke an undesired

immune response.3/27/2016Hari Pankaj Vanam #Micro@ BMC


5. Production laborious and

expensive

Variation in batch!!,

Prod time: can-Wks to

months-subject to

contamination.

SELEX-Little to no

variation, cost effective

7 times less of mAbs

Automated,

Selected in Hrs.

no risk of contamination.

6.Target

Potential

Targets: must cause a

strong immune

response (epitops &

IR!!).

Targets: Do not cause much

IR

also bind to ions-molecular

recognistionUnlimited

applications-

7.Size: (~150kDa) large

molecules resist

filtration –kidneys-

longer half-lives. prevents access to

smaller areas!!

(8–25kDa )Small

molecules subject to

kidney filtration, shortened

half-lives. bind to smaller

targets that Abs cannot

reach.3/27/2016Hari Pankaj Vanam #Micro@ BMC


8.Ability to be

Modified

cannot accommodate

conjugates

chemically altering or

attachment, or

conjugation(PEG) to resist

renal filtration, drugs (i.e.

doxorubicin), toxins, &

siRNAs.

9.Patents and

Distribution

well-developed

infrastructure

no early patents

protected by patents until very

recently (IPT-18-20yrs )

Many labs are reluctant to

switch away from the

"tried,"

suppression of the

technology!?!

Aptamers are not the replacement of mAbs but an

attractive solution in scenarios where antibodies'

efficiency is limited 3/27/2016Hari Pankaj Vanam #Micro@ BMC

Aptamer modifications

A major problem small size –

susceptibility to renal filtration

nuclease degradation –

short blood half-lives ~ two minutes - a barrier to

in vivo therapeutics.

"disdvantages" turned into "advantages,“

natural disposal of aptamers –not retained

hence no negative side effects.

Can be modified to overcome above

shortcomings.3/27/2016Hari Pankaj Vanam #Micro@ BMC

increasing nuclease resistance.

boranophosphate /

phosphodiester backbone

Substitutions of the 2′-OH


Spiegelmers(1996): Biostable aptamers –Type of

SELEX locked nucleic acid technology or

generate “mirror” RNA results in structural

changes in RNA sequences resist nucleases.


RNA aptamer DNA Aptamer Peptide

Aptamer

15-60 nucleotide bases

SELEX

Form complex 2˚& 3˚

HAVE 2′-OH group and

non-Watson-Crick

basepairing allows

RNA aptamerdiverse

3D structures than

ssDNA.

15-60 nucleotide

bases SELEX

Less complex 2˚&

3˚

lack - 2′-OH groups

DNA naturally

resistant to 2′-R--

endonucleases/sta

ble

small, simple

peptides with a

single variable

loop region tied

to a protein on

both ends.

bind -targets with

variable loop

region

Reduces the

flexibility

Effectiveness

Upon recognition of their target, 3/27/2016Hari Pankaj Vanam #Micro@ BMC

RNA aptamer DNA Aptamer Peptide Aptamer

Combination 2˚ Structures 3˚ structures

entire aptamer folds into a stable complex

with the target ligand. (bind entire seq) via

vander Waals forces, H bonding and

electrostatic interaction – as Ab bind

Ags.

aptamer-target complex:

This 3D structure function like antibodies

not just information holding!!!

Structure

Constraint by

scaffold

Due to Post-

translational

modifications,

cannot fold into

the correct 3D

structure.

Require-reverse

transcription, in

which RNA DNA (

extra steps in

SELEX)

Inherently more

stable, cheaper,

and easier to

produce

All 3 aptamers have Biosensor,

Diagnostic, Therapuetic applications3/27/2016Hari Pankaj Vanam #Micro@ BMC

Process: SELEX An in vitro, or test tube-based combination of chemical reactions

that synthetically -isolate, rather then create(Mol recognition),

aptamers.

Systematic Evolution of Ligands : When mixed with

targetsNon-binding nucleotides are culled from the binding

nucleotides, in an essentially evolutionary process at a

molecular level.

Exponential Enrichment : exponential nature of the amplification

stage of aptamer selection.

3 Basic Steps:

1. Library Gen –random libray of 1013-1016 ssDNA or RNA

2. Binding Seperation– incubated with the target molecule.A few

NA bind to target -aptamers. Process of –Elution Unbound NAs

are filtered out of the solution.

3. Amplification– binding NA s copied PCR-Create new library.

used in a new round of SELEX to further optimize the quality of


5′-sense primer -random sequence -

fluorochrome reporter for monitoring

aptamer selection &-antisense primer

sequence-3′-labeled with an affinity

molecule, ex: biotin

reporter biotin

1. Library Gen

2. Binding Seperation

Partition & Elution

3. Amplification


Purified rProteins or peptides expressed in prokar/eukary.

Due to Post-translational modifications, ex: highly glycosylated proteins, purified proteins or peptides - cannot fold into the correct 3D structure.

This limits selectively recognize and interacting targets-hence choosing biomarkers in their native conformation MUST for aptamers selection.

We Shall discuss Cellbased SELEX (or Cell-SELEX)-

Widely used for Discovery of biomarkers:

Ex: protein tyrosine kinase 7 (PTK7) and Ig heavy mu chain -highly expressed in solid tumors and on lymphoma cells.

Well-characterized biomarkers –

ErbB superfamily, MUC1, EpCAM, and CD30.

Peptide Aptamers As BIOMARKERS


Whole cells- Discovery of biomarkers: presence of many different cell

surface molecules in addition to the target biomarker(s) synthesis of many

unrelated/unwanted aptamers- also utilizes control cells that do not express

the target biomarker(s) during the counter-selection step.

Lets synthesize DNA-aptamer specific for CD30

+ ve Selection

- ve Selection


DNA-aptamer specific for CD30, biomarker +++ Hodgkin and anaplastic

large cell lymphoma (ALCL)-exhibited high CD30 binding affinity as low as

2 nmol/l + stable in human serum for up to 8 hours.

Conversely, an RNA-based CD30 aptamer was digested within 10 minutes.

+ ve Selection- ve Selection


Timeline of emerging modification-SELEX


improve efficiency/optimization


exciting potential to treat specific diseases

conditions for which current methods have reached dead

ends.

1. Acting as an antagonist, -

protein-protein and receptor-ligand interactions.

2. Acting as an agonistactivating cellular receptors.

3. Acting as a drug-delivery agent ; target diseased

cells with pinpoint accuracy to deliver a drug dose.

Aptamer Applications


Aptamer Applications


Aptamer applications –1. HIV/AIDS

Model of potential RT binding site of HIV

1

RT-sort of "hand" The green"thumb“

section of the enzyme closes over the

genetic material when transcription is

underway.

Blue –fingers, Red for palm.

One School of thought: "Competitive

Inhibition." Aptamers compete with viral

RNA/DNA for the area of the "palm"

region of the RT enzyme that reverse

transcription occurs at.

The aptamer binds into the enzyme,

preventing its use by the virus –

therefore inhibiting viral replication.

Can become a "foolproof way to stop RT“!


Search for broad spectrum aptamers

For "foolproof way to stop RT“!

The ability to distinguish between the many HIV subtypes

within both type HIV-1 and HIV-2 & prevent RT from

structurally evading the aptamer.

Research is focused primarily on structural and functional

details of aptamer-RT interactions which pave wags to

develop and discover functional broad-spectrum RT inhibitor

aptamers. 3/27/2016Hari Pankaj Vanam #Micro@ BMC

2. Age-Related Macular Degeneration (AMD)

Deterioration of the Macula/Retina

@ Risk of vision loss.

In Wet AMD -abnormal vessel growth is

caused by VEGF. Normally, VEGF is

released to increase oxygen flow to the

eye,the blood vessels leak and

disrupt visionblind spots, loss of

central vision, and making straight lines

appear "wavy." The blood vessels

eventually scar, making vision loss

permanent.


Macugen –

First Ap-Based AMD Rx

Pegaptanib Sodium-MACUGEN targeting VEGF

resist nuclease degradation.

conjugated to PEG to increase size, slows tissue absorption and renal filtration - lengthening half-life.

Approved in Dec 2004 FDAmarketed by Pfizer & Eyetech.

Inhibits interactions between VEGF and its receptors, preventing VEGF from stimulating blood vessel growth.

Exisiting blood cells, without a continuing flow of VEGF, die off.

Administered in 0.3 mg doses every 6 weeks by intravitrealinjection - injection directly into the eye.

The aptamers travel through the eye, locate, and then bind to VEGF with the high specificity and affinity of aptamers.


Other potential application of Aptamers

Aptamers selected against bacteria can act as antibiotics

by inhibiting bacterial proteins or disrupting cell membrane

formation.

Can also be linked antibiotic agent, deliver the antibiotic.

Trypanosoma cruzi, :RNA aptamers bind to the parasite

receptors on the trypomastigote cell surface, and blocked cell

invasion by 50-70%.

Plasmodium parasite- Aptamers against parasite proteins

to disrupt the disease, eventually by supplement therapy for

severe malaria.

Prions: Aptamers targeting prions can distinguish readily

between abnormal and normal proteins. By binding to prions,

they can prevent the accumulation of prions - spongiosis.


Autoimmunity: Myasthenia gravis-antibody response to a receptor called the nicotinic acetycholine receptor (AChR). Animal models - RNA aptamer targeting these autoantibodies inhibits this autoimmune response.

Coagulation: used in surgery -prevent disastrous clotting.

Against thrombin- coagulation, to prolong clotting time in human blood plasma.

An aptamer targeting Factor IXa- coagulation, has entered Phase IIb clinical trials involving 800 patients with Acute Coronary Syndrome undergoing cardiac catheterization - or introduction of artificial tubes to clear blocked blood flow in clogged arteries. This aptamer is coupled with an "antidote" that reverses its effects.


Examples of RNA aptamers in clinical trials


Diagnoses of Infectious diseases

Aptamers directed against spores of Bacillus anthracis

vaccine strains Sterne- isolated and characterized.

Patents- aptamer chip, containing spore-binding

aptamers.

NSP 3 of HCV- HCV RNA replicase has been identified

in patient's blood using an RNA aptamer immobilized

on magnetic bead and analyzed by MALDI-TOF.

Tools for detection -Toxins: DNA aptamers directed

to the Cholera toxin and the Staphylococcal

Enterotoxin B (SEB) have been isolated-

Spiegelmers.

Dx brain diseases: Alzheimer's disease-High-affinity

RNA aptamers against the amyloid peptide

(correlated with)3/27/2016Hari Pankaj Vanam #Micro@ BMC

Dx cardiac diseases -RNA aptamer binding SAH (S-

adenosylhomocysteine Biomarker) with an affinity

and selectivity comparable to that of a mAb and with a

high diagnostic potential has been isolated.

Imaging: DNA aptamer binding to the enzyme

neutrophil esterase- in vivo rat model

Neoangiogenesis -25 Aptamers studied on - serve as

molecular addresses differentiating the tumour

vasculature eg: brain glioblastoma but not the

vasculature of the normal brain.


Aptamers in Analysis:

ALISA

Capillary electrophoresis &

affinity chromatography,

rapid determination of low

concentrations of IgE, thrombin

and HIV-1 RT.

Biosensing -Ap- Beacon

Diagnostics

aptamer-based fluorescent

reporters.


Aptamers in Cancer!!

Contemporary cancer treatments include surgery, chemoTx,

RT, PDT, and Photo Tx serious side effects in patients

due to their associated nonspecific toxicity.

Personalized, targeted therapy- employs antibody-based

drugs- fewer side effects, potential immunogenicity and

high cost of production may limit its clinical applications.

Aptamers being developed against in considering these –

four criteria-

1).Overexpression of the target antigen in tumor cells,

2).the same* -integral part of disease progression,

3).stable form on tumor surface, and is

4).expressed by a large % of tumor cells / variety of

tumors.3/27/2016Hari Pankaj Vanam #Micro@ BMC

APTAMER-MEDIATED TARGETED THERAPIES

Aptamer technology in

Personalized, targeted therapy-

offers numerous advantages

1.Aptamer-drug conjugates. (ApDC)

2.Ap-nanoparticle therapeutics

( ANPs) Theranostics!!!

3.Ap-mediated gene therapy

4.Ap-mediated immunotherapy

5.Ap-mediated target cell biotherapy/


CENTRAL PATHWAY OF FUTURE APTAMER BASED Personalized, targeted therapy.


1.Aptamer-drug conjugates (ApDC)

Noncovalently or covalently conjugating (most common)

aptamer sequences directly with therapeutic agents.

Ex: Aptamer-conjugated Doxorubicin vs Dox alone –

being used in treatment of various cancers-exploiting its

MOA intercalation into the nucleic acid structure inhibit

cancer cell proliferation with a incubation step.

Examples: ApDCRx of retinoblastoma


ApDC

2′-fluoro modified RNA aptamer EpDT3+Dox ((specific

for EpCAM, a CSC marker).

HER2 ApDC targeting breast cancer.

MUC1 ApDC targeting lung cancer, (PDT with ApDC)

PSMA ApDC targeting prostate cancer.

Gemcitabine (Gem is an FDA-approved

deoxycytidine analog (dFdC) Gem polymer for EGFR

Necleolin aptamer (AS1411breast cancer.

EGFR aptamer- Lung cancer/Pancreatic cancer.

Mono/Bi-/Trispecific Aptamer PSMA-positive and -

negative cell types


Towards Better ApDC

Better-ApDC drug delivery good half life-Releasing Dox

and effectively killing target cells.

Ex: DNA aptamer sgc8 strong affinity for PTK7 kinase

highly expressed - Acute lymbhoblastc leukemia.

Increase aptamer Mw with polymers - (PEG) PEGylated

aptamers. Ex: Macugen aptamers prolonged its

circulating half-life, but also enhanced its stability and

decreased its toxic accumulation in non target tissues.


sgc8 aptamer- NTrs-Dox conjugates were

preferentially internalized by the target cells-

Increasing payload capacity, inhibiting tumor cell

growth in vitro and in vivo.

Other strategies-construction of polyvalent aptamers

First, dimeric aptamer complex (DAC) specific

delivery of Dox to PSMA-expressing cancer cells.

Polyvalent aptamers(Mono/Di/Tri) constructed through

the rolling circle amplification (RCA) technology-

killing leukemia cells and prostate cancers PSMA-

positive and -negative cell types are seen.

Aptamer-tethered DNA nano-trains (aptNTrs)

Theranostics


2. Ap-Nanoparticle therapeutics (ANPs)

Functionalized copolymers or liposome NPs are an

effective, universal, and safe platform for targeted drug

delivery enhanced biodegradability and biocompatibility.

Aptamer-NPs bioconjugates Farokzad etal

Theranostics


Copolymers and liposomes:

aptamer-NPs bioconjugates

ex: anti-PMSA (target)+PLA/PEG/PGLA (Control

DR)

Circulating half-life of the resultant bioconjugate-

Docetaxel (Dtxl) loadingreduced systemic toxicity

and a significant in vivo tumor burden reduction.

Many aptamer-NPs bioconjugates developed

Ex: MUC1 adenocarcinomas

PLGA-lecithin-PEG and PTX-containing AS1411

aptamer-breast cancer therapy


Metal nanomaterials

Apt-HAuNS-Dox synthesis.

Aptamers and PEG were conjugated to the surface of HAuNSsequentially via covalent S-Au bonds, loading with doxorubicin.

selectively kill lymphoma cells, but exhibit no cytotoxicity in other cell types.(In vivo cell lines)

Other strategies include; gold nanomaterials in PTT/PDT

(SPION) Super paramagnetic iron oxide nanoparticles imaging detection and treatment 3/27/2016Hari Pankaj Vanam #Micro@ BMC

VLP and other nanomaterials-ANPs

VLP’s- Naturally derived

nanomaterials with

multifunctional properties -

drug delivery.

Ex: sgc8 aptamer were

conjugated on the surface of

each MS2 bacteriophage

capsid.

Other Nanomaterials such as hydrogels, silica,

quantum dots (QD), and single-walled carbon

nanotubes are also promising vehicles for aptamer-

mediated delivery of targeted therapeutics -

successfully used to enhance biodistribution, stability, and

targeting affinity of aptamers 3/27/2016Hari Pankaj Vanam #Micro@ BMC

3.Aptamer-mediated gene therapy

Gene silencing tools -*cancergene-mediated

therapeutics.

Small interfering RNA (siRNA) and microRNA (miRNA)

Alone -their lack of cell/tissue specificity during in vivo

delivery.

when covalently conjugated with aptamers to form aptamer-

siRNA or aptamer-miRNA chimeras.

Using siRNA/miRNA technology + aptamers-acheives

selective gene targeting with high efficiency.

HIV research: siRNA aptamer against gp120 was covalently conjugated with a

tat/rev siRNA.Anti-gp120 aptamer-siRNA chimera inhibited HIV replication

and host-to-host spread. 3/27/2016Hari Pankaj Vanam #Micro@ BMC

Nanocomplex for ALCL cells. Anaplastic Large Cell Lymphoma.

functional nanocomplex is added to cultures, the aptamer component will

selectively target CD30-positive ALCL cells will facilitate intracellular

delivery of the nanocomplex.

The siRNA component will subsequently silence the cellular ALK gene,

resulting in the growth arrest of ALCL cells.

RNA-based CD30 aptamers-Gene Silencing

ALK siRNAfunctional


4. Aptamer-mediated immunotherapy

Adoptive immunity: low potential side effects and high specificity. Through the Fc functional region ADCC, complement-mediated cytotoxicity, and phagocytosis/opsonization.

Chimeras, Bi & Multi specific aptamers all have potential therapeutic applications especially as adoptive immunotherapy.

Stecker et al. Chimera :aptamer-C1q conjugate-

MUC1 aptamer +C1q molecule through a biotin-streptavidin connector- killing effect using the MCF7 breast cancer cell line therapeutic effectiveness.

Boltz et al bi-specific c-MET-CD16α Aptamerpromoted recruitment of the natural killer cells and induced killing of the target cells.


5.Aptamer-mediated target cell biotherapy

Cell surface receptors-Involved -biological processes-

viz: signaling transduction

An abnormal expression related to tumorigenesis.

60% of cancer-targeting drugs- target cell surface

biomarkers.

advancement of both the protein- and/or cell-based

SELEX technologies attractive for disease

treatment.

extensively studied for diagnosis and/or treatment

of hematological malignancies, lung, liver, breast,

ovarian, brain, colorectal, and pancreatic cancers,

as well as for identification and characterization of

CSCs.(Car Stem cell) 3/27/2016Hari Pankaj Vanam #Micro@ BMC

Ap + phosphorothioates, selectively bind to E-selectin that is presented on

the surface of human endothelial cells. The aptamers serve as antagonist

for the interaction to sialyl Lewis X on HL-60 cells, a human promyelocytic

leukemia cell line, thereby inhibiting the adhesion of tumor cells by more

than 75%.

Aptamers as

blocking agents.


-aptamers confer agonistic or

antagonistic effects on their

specific biologic functions-

cancer cell death.

Mono Vs Poly:

Monovalent aptamers, unable to

activate the downstream signaling

pathways.

Conversely-multivalent aptamer

induce receptor multimerization,

thus triggering downstream

signaling.


Conclusion

Aptamar research is Expanding -viable clinical agents.

Unique class of molecules pKhence exploited.

Nonimmunogenic ,and amendable -modification and

conjugate with nanoparticles, imaging agents, siRNAs

and therapeutic drugs.

Ligands for treating /Dx cancer, HIV, and other diseases.

Able to form more stable 3D structures due to the strong

intrastrand RNA-RNA quicker selection but multiple steps.

Yet DNA aptamers have better chemical and

thermodynamic stability -B helix.


RNA aptamers cost constraints ex: the cost of 2′-fluoro,

2′-ribo & 2′-methyl RNA phosphoramidites ~$20 per gram.

Increased demand or new synthesis- reduce these

costs.

Difficulty to synthesize longer modified aptamers.

Advances in nucleotide synthesis technology will

likely also overcome this .

Cutting edge technology for 21st century -therapuetic

and Diagnostic use!!Their rapid development -last two

decades!!-we anticipate that they will become a

standard tool for clinicians in the near future.

Beginning of new era of chemical abs- potential to

compete mAbs in all aspects. 3/27/2016Hari Pankaj Vanam #Micro@ BMC

References:

1.http://aptamerstbc2013.wix.com/aptamers#!a

pplications/cfvg

2. Molecular Therapy—Nucleic Acids (2014) 3,

e182; doi:10.1038/mtna.2014.32 © 2014 The

American Society of Gene & Cell Therapy All rights

reserved 2162-2531/14

3. Ni et al. Curr Med Chem. 2011 ; 18(27): 4206–4214.

Thanks for WWW…..its purely educational a part of

PG Training.


http://aptamerstbc2013.wix.com/aptamers#!applications/cfvg

Profusely

Our Department

for giving me this

opportunity…


Aptamer Hari pankaj vanam aptamer a plethora of opportunities

Health & Medicine

Transcript of Aptamer Hari pankaj vanam aptamer a plethora of opportunities