Triple helix forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product as in the antisense strategy. However, the poor stability of some of these structures might limit their use under physiological conditions.
Genetic alterations in sporadic triple negative breast cancer.
Specific ligands can intercalate into DNA triple helices and stabilize them. This review summarizes recent advances in this field while also highlighting major obstacles that remain to be overcome, before the application of triplex technology to therapeutic gene repair can be achieved.
Triple helix formation fig. In intermolecular structures, an oligopyrimidine-oligopurine sequence of DNA duplex is bound by a third-strand oligonucleotide in the major groove [ 3 ].
Figure 1: DNA triple helix Two main types of triple helices have been described, depending on the orientation of the third strand [ 4 ].
Genetic alterations in sporadic triple negative breast cancer. - 中国知网
The first reported triple-helical complexes involved pyrimidic third strand whose binding rests on Hoogsteen hydrogen bonds between a T-A base pair and thymine, and between a C-G base pair and protonated cytosine [ 56 ].
The T, C -containing oligonucleotide binds parallel to the oligopurine strand in the so-called pyrimidine motif. A second category of triple helices contains purines in the third strand, which is oriented antiparallel to the oligopurine strand. Oligonucleotides containing Cancer genetic mutations and G can also form triple helices whose orientation depends on the base sequence [ 9 ].
Triple helix formation offers a direct means of selectively cancer genetic mutations gene expression in cells where DNA triple helices offer new perspectives toward oligonucleotide directed gene regulation fig.
Synthetic triple helix forming oligonucleotides TFOs bind with high affinity and specificity to the purine strand in the major groove of homopurine- homopyrimidine sequence in double-stranded DNA [ 10 ]. They have been studied in antisense applications, where they are designed to target mRNAs, antigene applications, where they control gene expression via triple helix formation, and in applications that target proteins, where they are used as aptamers [ 112 - 15 ].
TFOs can condiloame pe tot corpul be used in gene therapy where they target DNA sequence cancer genetic mutations mutated gene to prevent its transcription.
Triplex-mediated modulation of transcription has potential application in therapy since it can be used; for example, to reduce levels of proteins thought to be important in disease processes.
TFOs can also be used as molecular tools for studying gene expression and they have been proved to be effective in various gene-targeting strategies in living cells. The specificity of this binding raises the possibility of using triplex formation for directed genome modification, with the ultimate goal of repairing genetic defects in human cells.
Several studies have demonstrated that treatment of mammalian cells with TFOs can provoke DNA repair and recombination, in a manner that can be exploited to introduce, desired sequence changes.
A number of studies have been reported in which oligonucleotides were utilized as antigene compounds within the cells [ 2 ]. Formation of triple helix DNA Triple helix formation is a result of oligoinucleotides binding with a high specificity of recognition to the major groove of double helical DNA by forming Hoogsteen type bonds with purine bases of the Watson-Crick base pairs, the compound rationally designed for artificial regulation of gene expression [ 16 ].
In the triple helix or antigene strategy, the oligonucleotide binds in the major groove of double-stranded DNA via Hoogsteen hydrogen bonding to form a triple helix [ 56 ]. There are four structural motifs for triplex formation that have been described based on the third-strand composition and its orientation relative to the purine-rich strand of the duplex.
The antiparallel orientation is favored by a greater number of steps, while a low number cancer genetic mutations steps favor the parallel cancer genetic mutations [ 18 ].
Once the best motif for binding a particular target sequence is established, problems with natural phosphodiester oligonucleotides limit the success of the antigene approach and the therapeutic applications of oligonucleotides in general. Oligonucleotides with the natural phosphodiester backbone are susceptible to endo and exonucleases.
The predominant activity that degrades oligonucleotides is 3'-exonuclease activity, but endonuclease activity has also been observed in some settings [ 1920 ]. Thus, for application as therapeutics in vivo TFOs must be able to resist both exonuclease and endonuclease activity in order to reach their target. A backbone modification that confers nuclease resistance but allows binding to double-stranded DNA with high affinity is required for the in vivo applications of TFOs.
Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents [ 2122 ]. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six-membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage [ 23 ] fig.
Morpholino oligonucleotides are resistant to enzymatic degradation [ 24 ] and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H cancer genetic mutations 2526 ].
They have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study cancer genetic mutations several of these methods found that scrape-loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells [ 27 ].
A recent report demonstrated cancer genetic mutations formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium [ 28 ].
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Figure 3: Structural presentation of phosphodiester DNA and morpholino Cations have been shown to play an important role in triple helix formation. When phosphodiester oligonucleotides are used as TFOs, magnesium is generally required for triplex formation with purine and mixed motif TFOs; it also speeds the reaction and stabilizes the triplex formed with pyrimidine motif TFOs cancer genetic mutations 29 - 31 ].
Other divalent cations have been shown to function in the same capacity as magnesium cancer genetic mutations regard to triplex formation [ 32 ]. Magnesium occurs at a concentration of ~0. Potassium occurs in the cell at a concentration of ~ mM, and at 4 mM in the blood.
High concentrations of potassium can inhibit triplex formation with guanine-rich oligonucleotides designed as TFOs by favoring other secondary DNA structures, such as dimers and quadruplexes [ 1834 - 38 ].