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BIOPHARMACEUTICS OF ANTISENSE MOLECULE AND APTAMERS

This document discusses antisense oligonucleotides and aptamers. It defines antisense molecules as synthetic DNA or RNA segments designed to bind mRNA and block protein production. Aptamers are single-stranded nucleic acids or peptides that bind targets with high affinity. The document outlines the mechanisms of antisense activity including RNase H degradation and translation blocking. It also describes SELEX, the process used to produce aptamers, and their potential applications in research, diagnostics and therapeutics. Finally, it discusses challenges and strategies to improve aptamer properties and utilization.

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PREENTED BY NOMRATA DAS M.PHARM 2nd sem Roll.no 8 GIPS
CONTENTS  ANTISENSE INTRDUCTION  ANTISENSE THERAPY  MECHANISM OF ANTISENSE ACTIVITY  ROLE OF ANTISENSE, APPLICATION ,CHALLENGES  ADVANTAGES AND DISADVANTAGES  APTAMERS INTRODUCTION  CLASSIFICATION  APTAMERS TARGET BINDING MECHANISM  PRODUCTION OF APTAMERS (SELEX)  PROPERTIES AND APPLICATION  ADVANTAGES AND DISADVANTAGES  STRATEGIES TO OVERCOME APTAMER LIMITATIONS  CONCLUSION
ANTISENSE MOLECULE • Antisense molecules are synthetic segments of DNA or RNA, designed to mirror specific messenger RNA (mRNA) sequences and block protein production. •Messenger RNA (mRNA) is single-stranded sequence of nucleotides is called "sense" because it results in a gene product (protein). • One way to target the genetic material is to block mRNA by using “antisense DNA”, which prevents the message from ever becoming a protein.
Antisense therapy •Antisense therapy is a form of treatment for genetic disorders or infections. • Antisense drugs blocks abnormal disease related proteins and are being researched to treat a variety of diseases such as cancers, diabetes, asthma, arthritis.
Protein production occurs in two phases Transcription and Translation. In the transcription phase , the DNA strand is used as a template for manufacturing an mRNA molecule. mRNA is responsible for communicating the genetic message in the DNA to the cell so that protein production can take place. In the translation phase , the mRNA travels to the ribosome, and carry out protein synthesis.
BASIC CONCEPT
ANTISENSE OLIGONUCLEOTIDES •Synthetic short segments of DNA or RNA are referred to as oligonucleotides (ODNs). •An antisense oligonucleotide is a single strand of nucleic acid or nucleic acid analogs. AS-ONs usually consist of 15– 20 nucleotides, which are complementary to target mRNA. •In this technique Short segments of single stranded DNA called Oligodeoxynucleotides are introduced in to the cell. •These Oligonucleotides are complementary to the mRNA, and physically bind to it.
•When these oligonucleotides combined with target mRNA, a DNA/RNA hybrid is formed, which is degraded by the enzyme RNase H.
MECHANISM OF ANTISENSE ACTIVITY
RNase H •RNase H is a non-specific endonuclease, catalyzes the cleavage of RNA via hydrolytic mechanism. •The RNase H-dependent oligonucleotides, which induce the degradation of mRNA . •The target mRNA is degraded, whereas the antisense oligonucleotide remains intact. Blocking Translation •When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked. This may occur because 1. The ribosome cannot gain access to the nucleotides in the mRNA 2. Duplex RNA is quickly degraded by ribonucleases in the cell.
ROLE OF ANTESENSE •To inactivate ethylene synthesis. • Reduction of starch content in vegetables. • Modification of flower colour in various decorative plant. • Mammalian genomes encode numerous natural antisense transcripts, but the function of these transcripts is not well understood.
APPLICATIONS  Genetic Disorder  Cancer  Renal Transplant Rejection  Infection (Viral, Protozoal, Fungal)  Cardiovascular disorder ( hypertension)  Inflammatory and autoimmuno disorders (Asthema) CHALLENGES • Need to identify appropiate gene target. • Need to generate antisense oligonucleotide that is specific for the gene of interest • Molecules need to be modified to prevent degredation by RNAses and other enzyme. • Drugs need to be deliverable to the organ of interest.
ADVANTAGES  Sensitivity of therapy can easily measured  Potential to produce long lasting responsrs  Oligonucleotides are manufactured quickly i.e, within a week. DISADVANTAGES  Completely artificial molecule, so no vectors possible  Not always highly effects.  Side effects for high dosage.  Delivery usually only via microinjection or electropration.
APTAMERS In, Latin aptus/apto – to fit, and In, Greek meros – part (Smallest Unit Of Repeating Structure) • Single stranded folded oligonucleotides and peptide that bind to molecular (protein) targets with high affinity and specificity • Provide opportunities for structure-based drug design strategies relevant to therapeutic intervention. •Can be prepared using SELEX. •They are similar to monoclonal antibodies.
CLASSIFICATION OF APTAMERS BASED ON STRUCTURE APTAMERS NUCLEIC ACID RNA AMINO ACID XNA DNA AFFIMER PEPTIDES
RNA aptamer DNA aptamer Peptide aptamer From complex secondary and Tertiary structure From complex secondary and Tertiary structure Structure constrains by Scaffoled (temporary structure 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, DNAAND PEPTIDE APTAMERS:
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. •Aptamers can binds to nucleic acid, proteins and small organic compounds APTAMERS – TARGET BINDING MECHANISM
APTAMER PRODUCTION SELEX • Systematic Evolution of Ligands by Exponential Enrichment •Aptamer are produced by an in vitro selection or systematic evolution of ligand by exponential enrichment (SELEX). • SELEX produces ssRNA with specific binding target. • This is an iterative process of binding, partitioning, amplifying novel nucleic acids & regeneration.
In the first step (library generation) : • A library, except the initial compound in the library, is converted into single- strand nucleotides that consist of random sequence regions, usually 30–40 meters, flanked by the primer binding site. In the second step (binding and separation) : • The target-bound library components are separated from the unbound components. This step is generally coupled with several other methods to make selection of the target or the library easy and rapid. Finally, in the third step (amplification) : • The target-bound library component is amplified by the PCR to create a new library to be used in the next round. Aptamers are continuously developed through this on-going process, and their characteristics are identified using various biological assays.
APTAMER SELECTION METHOD BASED ON SELEX 1. Nitrocellulose Membrane Filtration - Based SELEX 2. Affinity Chromatography & Magnetic Bead – Based SELEX 3. Capillary Electrophoresis-based SELEX 4. Microfluidic -based SELEX 5. Cell-SELEX
PROPERTIES OF APTAMERS • Ability to bind to proteins with high affinity and specificity. • 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, • 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.
APPLICATION OFAPTAMERS •For new drug •In drug delivery •As tehrapeutic tool •In diagnosis of disease •In food inspection •In hazard detection •In bio-imaging •As analytical reagent Aptamers have similar affinities
ADVANTAGES •Produced chemically in a readily scalable process • Not prone to viral or bacterial contamination • Non-immunogenic • more efficient entry into biological compartments • greater stability • Can usually be reversibly denatured. • Dyes or functional groups can be readily introduced during synthesis. •Aptamers have similar affinities as antibodies to target molecules. • They are non-toxic and elicit little or no immunogenic response.
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.
STRATEGIES TO OVERCOME APTAMER LIMITATIONS • Aptamers can be optimized for activity and persistence under physiological conditions during selection or during structure– activity relationship and medicinal chemistry studies conducted after discovery. • Addition of conjugation partners such as polyethylene glycol or cholesterol can increase circulating half-life. • Chemical modifications incorporated into the sugars or internucleotide phosphodiester linkages enhance nuclease resistance.
CONCLUSION Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention. Aptamers’ entire selection process can be carried out in a test tube. They have high binding affinities, selectivity and specificity to their targets making them important for diagnostics and targeted therapy. They are thermostable and can be structurally modified to enhance specificity and stability, therefore increasing aptamer half-life in in vivo applications.
References • http://en.wikipedia.org/wiki/Antisense_RNA •http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Antisens eRNA.html •Walton, S. P., Roth, C. M., Yarmush, M. L. “Antisense Technology.”The Biomedical Engineering Handbook: Second Edition. •Keefe, A. D., Pai, S and Ellington, A. 2010. Aptamers as therapeutics nature reviews, 9:537-50. •Mascini, M. Aptamers and their applications. Anal. Bioanal. Chem. 2008 •Gopinath, S.C. Methods developed for SELEX. Anal. Bioanal. Chem. 2007 •Tombelli, S.; Minunni, M.; Mascini, M. Analytical applications of aptamers.
BIOPHARMACEUTICS OF ANTISENSE MOLECULE AND APTAMERS