Antisense oligonucleotides and their mode of action

The nucleotide sequence of an mRNA molecule contains the encoded blueprint that dictates the amino acid sequence of a protein. Because of this, the mRNA sequence is said to make 'sense'. (This mRNA, therefore, is complementary to an 'antisense' DNA strand, i.e. it is the antisense strand of DNA in a given gene that serves as template for the mRNA synthesis.) As long as at least part of the nucleotide sequences of any mRNA is known, it becomes potentially possible to synthesize chemically an oligonucleotide, either a ribo- or deoxyribo-nucleotide, whose base sequence is complementary to at least a section of the mRNA sequence. As long as such an ' antisense' oligonucleotide can enter the cell, the complementarity of sequences can promote hybridization between the mRNA and the antisense oligonucleotide (Figure 14.14).

Successful binding, however, does not depend alone upon Watson-Crick base complementary. It is also influenced by higher-order secondary and tertiary structures adopted by the RNA.

Antisense oligonucleotide

Antisense oligonucleotide

Figure 14.14 Outline of how an antisense oligonucleotide can prevent synthesis of a gene product by blocking translation. In practice, antisense oligos are 12-18 nucleotides in length. In many instances, antisense binding is believed to occur in the nucleus

Intramolecular complementary base pairing can occur (particularly within transfer and ribosomal RNA, but also messenger RNA), resulting in the formation of short duplex sequences, separated by stems and loops. Such higher-order structure seems to be functionally important, conferring recognition motifs for proteins and additional nucleic acids, as well as helping to stabilize the RNA. Regions engaged in intramolecular base pairing are obviously poor targets for antisense oli-gos. It is thus desirable to synthesize a nucleotide whose sequence is complementary to an accessible sequence along the mRNA backbone. Various approaches are taken to identify such suitable sequences (remember, the entire sequence of the mRNA will be known). The 'blind' or 'shotgun' approach entails synthesizing large numbers of oligos targeted to various (often overlapping) regions of the mRNA. The ability of each oligo to block translation of the mRNA is then directly assessed in an in vitro assay system using cell-free extracts. The second design approach entails the use of various computer programs to interrogate the mRNA sequence in an attempt to predict its higher-order structure (and hence identify accessible sequences). This approach remains to be optimized. The translation initiation sites of mRNAs are often popular targets because they are essential to translation and they are generally free from secondary structure. However, sequence homologies can exist within these sequences in unrelated genes. This reduces the specificity of the blocking effect and could lead to clinically significant side effects.

Binding results in the blocking of translation of the mRNA and, hence, prevents synthesis of the mature gene's protein product. The prevention of mRNA translation by duplex formation with antisense oligonucleotides appears to be underpinned by various mechanisms, including: (a) the oligonucleotides act as steric blockers, i.e. prevent proteins involved in translation, or other aspects of mRNA processing, from binding to appropriate sequences in the mRNA; (b) the generation of duplexes also likely allows targeting by intracellular RNases such as RNaseH. This enzyme is capable of binding to RNA-DNA duplexes and degrading the RNA portion of the duplex (most synthetic antisense oligonucleotides are DNA based).

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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