ANTISENSE TECHNOLOGY AND ITS APPLICATION
The tool that is used for the inhibition of gene expression is called Antisense technology. The antisense nucleic acid sequence base pairs with its complementary sense RNA strands and thus prevents it from being translated into a protein. The complementary nucleic acid sequence can be either a synthetic oligonucleotide, like oligodeoxyribonucleotides (ODN) having less than 30 mucleotides or longer antisense RNA (aRNA) sequences (Sczakiel, 1997). Example of sense and antisense RNA is: 5’ A C G U 3’ mRNA, and 3’ U G C A 5’ Antisense RNA.
Dr. Hal Weintraub first developed this technology at Basic Science Division. Firstly, they showed that aRNA inhibits the gene expression in mouse cells by Berg, 2002. Dr. Meng-Chao Yao in 1996 showed that aRNA that was incorporated into non-conserved regions of ribosomal RNA (rRNA) disrupts translation and this was done by altering interaction of the mRNA, and the rRNA, mRNA chimera.
Sequence transcription of antisense DNA strand into the sense mRNA strand, which is then translated into polypeptide (Kimball, Nov 2002).
The inhibition in which the theory works are as follows:
When the RNA binds to the complementary mRNA, it forms a double stranded RNA (ds RNA) complex which is similar to double stranded DNA . The dsRNA complex do not allow translation to occur. This translation process was not known.
Several theories include:
- dsRNA prevents ribosome from binding to the sense RNA and translating.
- dsRNA cannot be translated from nucleus to cytosol, where the translation occurs.
- dsRNA is susceptible to endoribonucleases that does not affect single stranded RNA, but degrade the dsRNA.
Oligonucleotide-based antisense techniques represents the most successful approach to genetic message element. Zamecnik and Stephenson first demonstrated the antisense effect of synthetic nucleotide. Zamecnik and Stephenson identified a repeated sequence of 21 nucleotides (nt) that was crucial to viral integration with the help of nucleotide sequences from the 5’ and 3’ ends of the 35S RNA of Rous sarcoma virus (RSV). They synthesized a 13-mer oligonucleotide, d(AATGGTAAAATGG), complement to the portion of this viral sequence. Viral production got inhibited when synthetic oligonucleotide was introduced into cultured fibroblast cells.Thus, they concluded that oligonucleotide was inhibiting viral integration by hybridizing to the crucial sequences and blocking them. They introduced the term ‘hybridon’ to describe such oligonucleotides.
At the same time, Tennant et al and Miller et al reported similar effects for synthetic oligonucleotides in other systems. These results focused on the ability of synthetic oligonucleotides to interfere with gene processes. Synthetic oligonucleotides are foreign to the cells into which they are introduced and thus becomes prey for endogenous nucleases. Synthetic oligonucleotides were protected from endogenous nuclease when they attained the persistence level in cell. There are three possible sites on a nucleotide where protective modifications could be introduced.
The three possible sites are Base, Ribose (2’ OH group) and the Phosphate backbone.
In RNA nucleotides the 2’ hydroxyl group, missing in DNA nucleotides, can be modified. The alteration was thus introduced in the protective modifications of nucleotides that protects against the nuclease degradation which does not at the same time eliminates the desired effect of the oligonucleotide sequence by blocking the complementary hybridization or harming of the cells.
In the late 1960s, Eckstein and colleagues successfully introduced the first-generation antisense-motivated nucleotide modification. They replaced one of the non-bridging oxygen atoms in the phosphate backbone with a sulfur atom. This modification was called as phosphhorothioate that achieved the goal of nuclease resistance measured by an increased half-life for a phosphorothioated oligonucleotide upto ten hours in human serum as compared to that of one hour of an unmodified oligonucleotide having the same sequence. Moreover, Matsukura and colleagues demonstrated that phosphorothioated oligonucleotides were effective hybridons against the HIV replication in the cultured cells. On the other hand, phophorothioated oligonucleotides displayed slightly reduced hybridization kinetics and a tendency towards unspecific binding with certain proteins which resulted in cytotoxicity at high concentrations. Thus, the dose-response was added to the mix of issues for antisense agents and hence the useful modifications continued.
The so called second-generation class of modifications directly addressed the non-specific and cytotoxic issues which was raised by the phosporothioates by the introduction of RNA oligonucleotides with alkyl modifications at the 2’ position of the ribose sugar. The two most important of these modifications are 2’-O-methyl and 2’-O-methoxy-ethyl RNAs. Antisense nucleotides contains these modifications and displayed the nuclease resistance in concert with lower toxicity and major drawback of 2’-O-alkyl modifications is that the antisense agents containing them are unavailable to the most powerful antisense mechanism called RNase H cleavage. Steric block mechanism are affected from these agents. Thus, the 2’-O-methyl oligonucleotides have been used to increase the desired expression of alternate splices in certain proteins by suppressing the undesired splice variant.
Since RNase H cleavage is the most desirable mechanism for antisense effect. Nuclear resistance rarely have 2’-O-alkyl modifications which is a hybrid oligonucleotide, constructs incorporating both the characteristics has been appeared in the form of the “gapmer” antisense oligonucleotide, containing central deoxynucleotide blocks sufficient to induce RNase H cleavage which was flanked by the blocks of 2’-O-methyl modified ribonucleotides, thus protecting the internal blocks from nuclease degradation and these irrelevant cleavage appears because of binding short stretched nucleotide in most of the genomes. For example, a 15-mer can be viewed as a series of eight overlapping 8-mers. mRNA has less potential random targets, while in RNase H cleavage it is still high. This theoretical potential became real in the case of 20-mer phophorothioate oligonucleotide targeted to the 3’-untranslated region (UTR) of the protein kinase C alpha gene (PKCα).
While unmodified oligo-deoxynucleotides forms desired DNA:DNA and DNA:RNA duplexes. A variety of nucleic acid analogs have been developed by that increased the thermal stabilities when hybridized with the complementary DNAs or RNAs as compared to unmodified DNA:DNA and DNA:RNA duplexes. These are third generation antisense oligonucleotide modifications and the analogs are: peptide nucleic acids, 2’-fluro N3-P5’-phosphoramadites, 1’, 5’-anhydrohexitol nucleic acids, and locked nucleic acids.
The newest and most promising third generation modification is the locked nucleic acid (LNA), introduced by Koshkin et al, Obika et al and Singh et al. LNA, is thus composed of locked nucleotides into a single conformation through a 2’-0’, 4’-C methylene linkage in 1,2:5,6-di-O-isopropylene-α-allofuranose. LNAs increased the thermodynamic stability and enhanced nucleic acid recognition.
Ribozymes are RNA enzymes were first described by Cech in Tetrahymena thermophilia. Antisense agents immediatley seized the RNA processing capabilities of these enzymes. Thus, the hammerhead ribozyme was characterized. This enzyme was first isolated from viroid RNA by Ulhenbeck and Haseloff and Gerlach.
RNA Interference (RNAi):
RNA interference (RNAi) was first described by Fire and colleagues in Caenorhabditis elegans. Long-double stranded RNAs were introduced into C. elegans. RNAi generated enormous interest by both those who view it as a potentially powerful antisense tool and recognize it like ancient eukaryotic cellular defense mechanism.
The overall goal in introducing an antisense agent into the cells either in vitro or in vivo is to suppress or completely block the production of the gene product. The normal transcription and translation is affected due to transition between DNA and amino acid sequence. DNA strand is transcribed into pre-mRNA at step one. In step two, through the action of 3 separate processes like 5’ capping, intron excision and polyadnelyation, pre-mRNA is converted into mature mRNA. In step three, transportation of mRNA is carried out in ribosomes into the appropriate poly-peptide.
The first target is transcription step to achieve antisense knock-down or knock-out, in which antisense agent is targeted to DNA itself, thus preventing transcription of the primary message. Dagle and Weeks noted that there are three ways in which this strategy can be carried out viz, minor groove binding polyamides, strand displaying PNAs, and major groove binding , triplex forming oligonucleotides. According to White at al, pyrrole-imidazole are minor groove binding polymers that achieve sequence-specific action through side-by-side pairing of pyrrole and imidazole amino acids. Less than 7bp appears in the target sequence of short stretched DNA. While PNA agents are longer and their mode of operation binds to complementary strand of DNA helix, displacing the complement. This process is thus aided by the fact that PNA:DNA duplexes are more stable than the DNA:DNA duplexes so that former is thermodynamically favored over the latter duplex. Triplex forming oligonucleotides have longer sequences and these agents create stable triplex DNA instead of binding to one of the DNA helix, while displacing the other helix of DNA. Both involves the interaction of TFO having purine bases in a polypurine:polyrimidine stretch of duplex DNA. Watson-Crick bonded is the target dsDNA sequence and triplex forming oligonucleotides binds to duplex through Hoogsteen hydrogen binding: T-A:T and C-G:C triplets. This strategy necessitates that only the purine-pyrimidine dsDNA can be targeted and the cytosine in TFO must be protonated. Thus the cytosine protonation is due to the requirement for the acidic conditions. Sorensen et al reported that the LNA containing TFOs stabilizes the triplex formation at physiologic pH. A 15-mer having seven LNAs raised the temperature for the triplex to duplex transformation from 33°C to ~66°C at pH 6.8.
The next level of antisense attack focuses on the processing of the pre-mRNA and the intron excision mechanism. In this process, the oligonucleotide-based agent is used. The sequence-specific binding of the oligonucleotide to the pre-mRNA is required to prevent intron-excision.
The antisense agent is then targeted to the mature rRNA and interferes with the transcription apparatus in either due to presence of the oligonucleotide which prevents formation of the ribosomal complex. In ribosomal complex, short RNA oligonucleotides are not stable due to presence of helicase enzymes, while longer RNA oligonucleotides activates RNAi pathway.
Finally the most used mechanism is that of the RNase H degradation of mRNA. RNase H is an endogenous enzyme which cleaves the RNA moiety of an RNA:DNA duplex. In both cytoplasm and nucleus, RNase H is found. During DNA replication it removes the primers of RNA from Okazaki fragments. The most powerful weapon assessing functions of gene is called RNase H activation antisense.
Kurreck thus listed 15 antisense oligonucleotides in total that are used in clinical trials against the diseases like cancer and asthama.
APPLICATIONS OF ANTISENSE TECHNOLOGY:
James Watson and Francis Crick proposed deoxyribonucleic acid, which consists of two deoxyribonucleotide molecules each having 5’and 3’end that defines a polarity for the DNA strand. After binding of these strands in antiparallel orientation 3’ of one being juxtaposed to 5’end of the other, they compose a complete DNA molecule. The two stands are bound together by pairing four complementary bases in each strand such that adenine present on one strand binds to second thymine, while cytosine binds to guanine in the second. Each strand contains all the genetic information called as mirror image. This structure permits transmission of genetic information by allowing a complementary strand produce for a single strand. One strand can therefore produce an entire DNA molecule, occurring during the cell division.
Each triplet set of nucleotides on a strand of DNA encodes an amino acid. In this process, one or another portion of one strand (a gene) is copied by ribonucleic acid polymerase II producing a compelementary molecule of ribonucleic acid, or RNA. This messenger RNA (mRNA) therefore, contains the same information which is contained in the gene that has been transcribed. Mature RNA molecule is left when the introns are excised. RNA molecule is thus exported to the cell, directing protein synthesis at ribosome. This takes place after intron excision and additional process.
Formulation of Antisense Technology:
DNA/RNA physiology is applied in various methods. Antisense technology is the most important application used. Here, oligonucleotide is introduced into cell which binds to its target mRNA through complementary based-pairing. This binding forms RNA dimer in cytoplasm and halts the protein synthesis.
Application of Antisense Technology invitro:
Antisense technology is used successfully in two general areas. The first one is fundamental research where antisense oligonucleotides introduced helps to determine the role of a specific gene. Cell growth and other changes occurred due to the production of angiotensin II. Cellular renin angiotensin system played an important role in variety of cardiovascular disorders like artherosclerosis and vascular hypertrophy.
Oligonucleotides were developed to inhibit the synthesis of angiotensin as it was difficult to demonstrate cellular system to be operative. It is a substrate form which the cells make angiotensin II. Cells make their own angiotensin II having growth promoting effects with the help of this technology.
Therapeutic Application of Antisense Technology:
Viral infections can occur when the antisense oligonucleotides are complementary to viral RNAs. Similarly, antisense oligonucleotides directed towards the oncogene product plays an important role to reduce growth of cancer cells.
The most widely used application of this technology is in gene therapy. In this case, a variety of vectors are used to introduce antisense encoding genes into larger number of cells in a patient or animal to produce long term inhibition of protein. For example, vectors having angiotensin II receptor sequences when introduced in animal models can cause long term normotension in hypersensitive animals.
- Damha, Masad. 2002 Oct 17. Making sense of Antisense.
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- Kurreck J. (2003) Antisense technologies: Improvement through novel chemical modifications. European Journal of Biochemistry, 270: 1628-1644.
- Miller PS, Braiterman LT, and Ts’ o POP. (1977). Effects of a trinucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, on mammalian cells in culture. Biochemistry, 16: 1988-1996.
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