Antisense for Drug Discovery

Antisense molecules are synthetic segments of DNA or RNA, designed to mirror specific mRNA sequences and block protein production. The use of antisense drugs to block abnormal disease-related proteins is referred to as antisense therapeutics. Synthetic short segments of DNA or RNA are referred to as oligonucleotides. The literal meaning of this word is a polymer made of a few nucleotides. Naturally occurring RNA or DNA oligonucleotides may or may not have antisense properties.
Antisense oligonucleotides are synthetic pieces of DNA (at least 15 nucleotides in length) that can hybridise to sequences in the RNA target by Watson–Crick or Hoogstein base pairing. An alternative antisense approach is the use of ribozymes that catalyse RNA cleavage and inhibit the translation of RNA into protein. Peptide nucleic acids (PNAs), which are DNA-like molecules, are potential antisense and antigene agents. Aptamers are synthetic chains of nucleotides that bind directly to target proteins, inhibiting their activity and are considered to be antisense compounds. A high-affinity DNA analogue, locked nucleic acid (LNA), confers several desired properties to antisense agents. LNA/ DNA copolymers exhibit potent antisense activity on assay systems.
1. Antisense Oligonucleotides for Drug Target Validation
Antisense technology uses genetic sequence information to design rapidly inhibitors of any gene target. Because of their exquisite specificity, antisense oligonucleotides can inhibit the selected gene only, without an impact on other closely related genes. As a result, antisense inhibitors allow the identification of function of that single gene target more precisely than any other method. Several companies have integrated antisense technologies in functional genomics.
DNA microarrays have been used to evaluate thousands of genes simultaneously. Target mRNA and protein expression can be inhibited using antisense oligonucleotides to facilitate this process and determine genetic pathways. Antisense technology is considered to be a viable
option for high-throughput determination of gene function and drug target validation.
Peptide nucleic acid (PNA) inhibits gene expression. If two different bacterial RNAs are targeted with a complementary PNA and a randomised control sequence, the complementary PNA inhibits its intended target, whereas the other does not. This provides an opportunity for novel antibiotic discovery in addition to target validation using antisense PNA. For validation of bacterial targets, the main advantages of PNA is it can be used for any bacterial species and the level of gene inhibition can be regulated. Genetic knockout is not applicable to all bacterial species and gene expression is an all-or-none phenomenon which cannot be regulated. Antisense PNA has the limitation that it cannot knockout 100% of the target whereas genetic knockout can do so. However, reduced levels of target activity mimic the therapeutic situation more realistically. Both techniques are synergistic and useful.In  conclusion, antisense PNA technology makes good sense for application in antibiotic target validation.
2. Aptamers
Aptamers (derived from the Latin word aptus=fitting) are singlestranded DNA or RNA oligomers, which can bind to a given ligand  with high affinity and specificity due to their particular 3D structure and thereby antagonise the biological function of the ligand. Aptamers are considered to be antisense compounds. The technology builds on the ability of aptamers to bind tenaciously to proteins and have been used to identify protein signatures. Streptavidin aptamers (streptavidin-binding RNA ligands) are also potentially powerful tools for the study of RNAs or ribonucleoproteins as a means for rapid detection, immobilisation and purification. Recent developments demonstrate that aptamers are valuable tools for diagnostics, purification processes, target validation, drug discovery and therapeutic.
Aptamer expression libraries do not depend on information from genome sequence. These can be used to find peptides with a desired biological activity. This can be caused by the expressed peptide activating or inhibiting a cellular factor. For stable expression of peptides in mammalian cells, libraries are constructed that express aptamers in the context of protease-resistant scaffold structures. A limitation of this approach is that highly complex mixtures of aptamers are required to identify active aptamers in any pathway. In addition, it can be cumbersome to identify the cellular factor that is affected by the biologically active peptide.
3. RNA as a Drug Target
RNA has a structural complexity rivalling that of proteins and thus provides an opportunity as a target for small-molecule drugs. Several steps are required to find and exploit RNAs as drug targets. Because all proteins are synthesised using RNA template, they can be inhibited by preventing the translation of mRNA. Advantages of targeting RNA are:
-Drugs that bind RNA can produce more selective action than those that bind proteins. For example, a drug can bind to a region of RNA which is relevant to the target tissue without affecting RNA in other tissues.
- Proteins are difficult to isolate or purify whereas RNA is easier to synthesise and use in assays.
- RNA can be easily synthesised in large quantities and is notext ensively modified in vivo, whereas large-scale production of proteins is still limited.
4 .Ribozymes
Ribozymes are enzymes comprised of RNA which can act both as a catalyst and as a genetic molecule. In Nature, ribozymes catalyse RNA cleavage and RNA splicing reactions and are sometimes called ‘catalytic RNAs’. Ribozymes are being increasingly used for the sequencespecific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeuticsare  a, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. For therapeutic purposes, a ribozyme can be considered to be a chimeric RNA molecule consisting of two stretches of antisense RNA flanking a nucleolytic motif. The antisense RNA component, referred to as the complementary flanking regions, provides target selectivity.
Unlike traditional pharmaceuticals, ribozymes disrupt the flow of genetic information rather than inhibit protein function. The therapeutic potential of ribozymes by targeting distinct mRNAs is tremendous and is a novel approach to curing disease. Diseases which result fromundesirable expression of RNA, such as neoplastic disorders and viralinfections , should be particularly amenable to this therapeutic approach. Use in genetic disorders is also being explored.