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Anti-miRNA oligonucleotides
Anti-miRNA Oligonucleotides (also known as AMOs) have many uses in cellular mechanics. These synthetically designed molecules are used to neutralize microRNA (miRNA) function in cells for desired responses. miRNA are complementary sequences (~22 bp) to mRNA that are involved in the cleavage of RNA or the suppression of the translation. By controlling the miRNA that regulate mRNAs in cells, AMOs can be used as further regulation as well as for therapeutic treatment for certain cellular disorders. This regulation can occur through a steric blocking mechanism as well as hybridization to miRNA. These interactions, within the body between miRNA and AMOs, can be for therapeutics in disorders in which over/under expression occurs or aberrations in miRNA lead to coding issues. Some of the miRNA linked disorders that are encountered in the humans include cancers, muscular diseases, autoimmune disorders, and viruses. In order to determine the functionality of certain AMOs, the AMO/miRNA binding expression (transcript concentration) must be measured against the expressions of the isolated miRNA. The direct detection of differing levels of genetic expression allow the relationship between AMOs and miRNAs to be shown. This can be detected through luciferase activity (biolumincescence in response to targeted enzymatic activity). Understanding the miRNA sequences involved in these diseases can allow us to use anti miRNA Oligonucleotides to disrupt pathways that lead to the under/over expression of proteins of cells that can cause symptoms for these diseases.
==Synthesis==
During anti-miRNA oligonucleotide design, necessary modifications to optimize binding affinity, improve nuclease resistance, and ''in vivo'' delivery must be considered. There have been several generations of designs with attempts to develop AMOs with high binding affinity as well as high specificity. The first generation utilized 2’-O-Methyl RNA nucleotides with phosphorothioate internucleotide linkages positioned at both ends to prevent exonuclease attack. A recent study discovered a compound, N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN), that improved binding affinity and blocked exonuclease degradation. This method was combined with the first generation design to create a new generation ZEN-AMO with an improved effectiveness.
Various components of AMOs can be manipulated to affect the binding affinity and potency of the AMO. The 2’-sugar of the AMOs can be modified to be substituted with fluorine and various methyl groups, almost all with an increase in binding affinity. However, some of these modified 2’-sugar AMOs led to negative effects on cell growth. Modifying the 5'-3' phosphodiester backbone linkage to a phosphorothiorate (P-S) backbone linkage was also shown to have an effect on target affinity. Using the P-S mutation was shown to decrease the Tm of the oligonucleotide, which leads to a lower target affinity. A final requirement for AMOs is mismatch specificity and length restrictions. Due to miRNAs in the same families sharing “seed” (shared) sequences and differ by only a couple of additional nucleotides; one AMO can potentially target multiple miRNA sequences. However, studies have suggested that this is difficult due to the loss of activity with single nucleotide mismatches. Greater than three mismatches demonstrates complete loss of activity. Changes in the length of AMOs were tolerated far better, with changes of one nucleotide and two nucleotides resulting in little loss of activity and three or more in total loss of activity. Truncating a single nucleotide from the 3’ end resulted in a slight improvement of AMO activity.
抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』
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