Antisense sequences

Unlike the situation with the integrins discussed earlier, it is loss of cadherin E that promotes metastasis. The evidence linking loss or decreased expression of cadherin E with cancer spread is as follows (M2). Firstly, a negative correlation exists between the expression of cadherin E and invasion for many different cancer cell lines. Secondly, in cell lines lacking cadherin E, invasion could be prevented by transfection with cDNA for this cadherin. Thirdly, reduction in cadherin E mRNA levels by antisense sequences induced the invasive phenotype in E-cadherin positive cells. Fourthly, antibodies inactivating cadherin E induced the invasive phenotype. These combined experiments are strong evidence that loss of cadherin E is associated with development of invasive phenotype and furthermore suggests that this adhesion molecule may be a suppressor of metastasis. 

Note that the complementary C nucleotide of the corresponding antisense sequences has to be omitted as well. 

RNA structures constitute one of the limitations to the use of the antisense strategy. Intramolecular basepairing competes with the intermolecular assocation between the antisense sequence and the sense RNA strand, and thus weakens or even abolishes the effect of the regulatory sequence. A number of studies have been devoted to the identification of accessible sites in target RNA (Sczakiel, 2000 Sohail and Southern, 2000) or to the optimization of the hybridization site for minimizing the penalty to be paid to unfold the target structure (Ecker, 1993 Freier, 1993). 

Mechanism of Action. Fomivirsen has a unique mechanism of antiviral action. This drug contains an amino acid sequence that is opposite or complimentary to the messenger RNA sequence controlling CMV replication. Because of this opposing or antisense sequence, fomivirsen inhibits several aspects of CMV replication, and also inhibits the viruses ability to adsorb to the surface of host cells. 

Generally refers to a nucleotide sequence that is complementary to a sequence of messenger RNA, which is the product of the noncoding sequence of DNA. It also refers to the peptide products from the antisense sequence referred to as antisense peptides. Antisense peptides have been investigated for biological activity. siRNA are based on the processing of antisense RNA. See Korneev, S. and O Shea, M., Natural antisense RNAs in the nervous system, Rev. Neurosci. 16, 213-222, 2005. See also MicroRNA, siRNA, Antisense Peptides, Aptamers. 

In addition to antisense targeting, hammerhead ribozymes have also been used to inhibit the expression of hTERT messenger RNA. These ribozymes are small catalytic RNA molecules that consist of a catalytic core flanked by antisense sequences that function in the recognition of the target sequence. These RNAs possess endoribonuclease activity that allows for the degradation of target transcripts. A hammerhead ribozyme has recently been used to cleave the hTERT mRNA in breast epithelial cells that inhibited telomerase activity and resulted in shortened telomeres, decreased net growth, and apoptosis (43). 

By reversing the polarity of the DNA strands, the opposite strand will be transcribed and a complementary or antisense RNA will be produced. When antisense RNA was introduced either in vitro (27) or in vivo (28), the presence of the antisense RNA inhibited the expression of the normal RNA transcript produced by the resident gene. Suppression of RNA translation by antisense RNA may be due to the formation of an RNA heteroduplex between the two complementary RNA strands thus blocking the attachment of ribosomes. An example of this application in the context of the Adh example would be the construction of a chimeric gene containing the antisense sequence for Adh linked with a female-specific promoter. Thus, Adh activity would be eliminated from females and make them lethally sensitive to treatment with ethanol. 

A strategy has been designed to minimize the number of dimers required for the synthesis of trimer phosphoramidite building blocks encoding all 20 amino acids. This requires a set of 7 different dimer blocks which can be used to synthesise all required antisense sequences. A set of 4 trimer phosphoramidites... 

The antisense sequence contains the complementary bases in an antiparallel arrangement. Thus, the definition of antisense compounds refers to their interaction with transcribed RNA. In a broader sense the term antisense action is also used to describe inhibition of mRNA processing by molecules binding to sites formed by tertiary structures of single-stranded mRNA and interaction of molecules with duplex DNA by sequence specific insertion into the grooves. 

Hammerhead ribozymes have been the preferred class of ribozymes to be used for in vitro experiments. In such experiments, the antisense sequence consists of 6-8 bases flanking both sites of the catalytic center. A lower number of bases would lead to decreased binding properties, while an increased number of pairing bases would reduce the turnover by decrease of... 

An increased tendency towards patents claiming antisense oligonucleotide application with specific mRNA-targets may be expected in future. According to expert opinion, such patents have a chance to be valid even in the case of previously patented cDNA-sequences. In this context, antisense-sequences and DNA-sequences may not be seen to be identical [448],... 

Fig. 2. Depletion of Mcl-l protein levels by antisense treatment. Neutrophils were reversibly permeabilised with streptolysin O and incubated with 20 iJuM carboxyfluorescein (CF), inverse antisense (Inverse) or antisense sequence 1 (AS) of chimeric oligodeoxynu-cleotides. Suspensions were incubated in either the absence (—) or presence (+) of GM-CSF. Western blots were analysed on aliquots of 10 cells, 4 h after permeabilisation. Actin (Ponceau S-stained blots) is shown to indicate equivalence of loading. Representative blot of 3 independent experiments. Antisense sequences and structures are given by Moulding et al. [28]. 

The thymidine oxyphosphorane 135 has been prepared by ester exchange, and underwent hydrolysis to give primarily the 5 -dialkyl phosphate. The cyclic analogue 136 of UMP has been synthesized for incorporation into antisense sequences to impart nuclease resistance. Its conformation was found to be similar to that of a nucleotide unit in A-type DNA duplexes. ... 

Finally, we determined the ability of the antisense sequence 2 to block the de novo HIV-1 infection in MT4 cells. In this experiment the a-RNA random sequence 4 was used as a control, and the corresponding a-DNA, a-S-DNA and 6-DNA sequences were also comparatively evaluated. 

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