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Synthesis of Nucleic Acid Foldamers and Analogs

RNA and DNA foldamers can be obtained by either chemical or enzymatic synthesis. DNA oligonucleotides of up to 120 nucleotides can presently be synthesized by phosphoramidite technology with reasonable yield [88]. However, the

Fig. 10.7 Examples of nucleotidomimetic foldamers. (a) Examples of carbohydrate-phosphate backbone modifications: LNA (Locked-in Nucleic Acid) [90], pRNA (pyranosyl RNA) [91], TNA (alpha-Threofuranosyl Nucleic Acid) [92], 5'-C-phosphononucleotide [93], PNA (Peptide Nucleic Acid) [94, 95]; (b) Examples of nucleotide linkage modifications:

phosphorothioate and phosphorodithioate linkages [96] and cationic guanidine backbone modification (deoxynucleic and ribonucleic guanidine) [97, 98]; (c) Examples of modifications at the 2'-OH position: 2'-O-Alkyl [10], 2'-amino [129] and 2'-fluoro [220, 221] modifications. These modifications have been incorporated in RNA and DNA aptamers (e.g. Refs. [221, 222]).

Fig. 10.7 Examples of nucleotidomimetic foldamers. (a) Examples of carbohydrate-phosphate backbone modifications: LNA (Locked-in Nucleic Acid) [90], pRNA (pyranosyl RNA) [91], TNA (alpha-Threofuranosyl Nucleic Acid) [92], 5'-C-phosphononucleotide [93], PNA (Peptide Nucleic Acid) [94, 95]; (b) Examples of nucleotide linkage modifications:

phosphorothioate and phosphorodithioate linkages [96] and cationic guanidine backbone modification (deoxynucleic and ribonucleic guanidine) [97, 98]; (c) Examples of modifications at the 2'-OH position: 2'-O-Alkyl [10], 2'-amino [129] and 2'-fluoro [220, 221] modifications. These modifications have been incorporated in RNA and DNA aptamers (e.g. Refs. [221, 222]).

lower coupling efficiency for RNA phosphoramidite synthons limits the length of single-stranded RNA foldamers to 45-50 nucleotides. Similar synthetic approaches have also been used to generate interesting nucleotidomimetic foldamers [89] with modified carbohydrates nucleotides such as 2 '-O-Alkylated RNA [10], LNA (Locked-in Nucleic Acid) [90], pRNA (pyranosyl RNA) [91], TNA (alpha-Threofuranosyl Nucleic Acid) [92] and 5 '-C-phosphono oligonucleotide [93], or modified backbone linkage like PNA (Peptide Nucleic Acid) [94, 95], phosphorothiates oligonucleotides [96] and cationic DNG and RNG analogs (deoxynucleic and ribonucleic guanidine) [97, 98] (Fig. 10.7). For an exhaustive review of nucleotido-mimetic foldamers before 2001, see the review by Moore and colleagues [89]. The advantage of the synthetic approach is that it can be used to create DNA/RNA analog hybrids or chimeric oligonucleotides and also allows the incorporation of a large variety of modified nucleobase analogs at precise locations within the oligonucleotide sequence [89]. It also offers access to DNA and RNA spiegelmers, single-stranded mirror images of DNA and RNA oligonucleo-tides that offer the advantage of being extremely resistant to DNA and RNA nucleases [99-101].

Alternatively, RNA and DNA of virtually any size and sequences can be obtained by in vitro enzymatic synthesis such as cloning [102], Polymerase Chain Reaction (PCR) or in vitro RNA transcription of plasmid and PCR generated templates [35, 103, 104]. Interestingly, a striking number of nucleotide triphosphate analogs are substrates for DNA and RNA polymerases, allowing enzymatic synthesis of DNA or RNA containing modification [7, 105]. In that case, the nucleo-tide analog is incorporated in the resulting transcripts at all the locations specified by the complementary template nucleotide. This approach can be used to investigate the structure and function of a RNA molecule by nucleotide analog interference mapping NAIM [106-108]. It has also been used to expand the functional scope of RNA or DNA by selection techniques (Section 10.4). The introduction of 2'-O-methyl and other substitutions into RNA and DNA can be facilitated by the selection and evolution of new polymerase variants that can incorporate modified nucleotides [109-114]. Interestingly, a great variety of chemically modified nucleotides with C5 amino-acyl groups can be incorporated during PCR by the KOD Dash DNA polymerase [115]. Until recently, the specific rules of natural DNA and RNA polymerases did not permit incorporation of a nucleotide analog at a specific and unique position within a RNA or DNA. However, the development of novel base pairings has recently been used to incorporate a site-specific fluorescent dye within an RNA, generated by transcription of a modified DNA template with 2-amino-6-(2-thienyl)purine triphosphate (sTP) or 2-amino-6(2-thiazolyl)purine triphosphate (vTP) in the presence of the modified complementary nucleotide triphosphate (2-oxo-(1H)pyridine triphosphate (yTP)) [116]. Alternatively, incorporation of site-specific modification within large RNA and DNA molecules is generally achieved by ligation strategies [117], thereby circumventing the size limitation of synthetic DNA and RNA. The yield of final products reached by these strategies is however still limited.

Particularly interesting are nucleotide analogs that can efficiently mimic the conformation of natural nucleotides helices (Fig. 10.7). For instance, LNA and 2 '-O-Me-RNA are both known to adopt A-form helix conformation and as such, are good structural analogs of RNA helices with chemically more stable backbones [10, 118]. For example, DNA/LNA chimeric oligomers have recently been shown to mimic RNA aptamers targeted to the TAR RNA element of HIV [119]. In this specific case, none of the 2 '-hydroxyls present at the level of the aptamers is critical for their function [119]. However, within large RNA sequences, the 2'-hydroxyls can be involved at key positions in 3° structure motifs and longrange interactions to promote folding and assembly into complex 3-D shapes. Nevertheless, it is possible to envision the synthesis of chemically more stable RNA foldamer analogs for which most of the ribonucleotide positions are substituted by chemically stable nucleotide analogs as long as these key 2 '-OH positions are known and kept unchanged.

It is also worth mentioning that synthetic and natural nucleic acids can be specifically modified by crosslinking [120-125], post-transcriptional modification at their 5' or 3' end extremities [126-128] or by specific modification of 2'-hydroxyl positions [129-131] in order to conjugate them with other molecular components.

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WHAT IT IS A three-phase plan that has been likened to the low-carbohydrate Atkins program because during the first two weeks, South Beach eliminates most carbs, including bread, pasta, potatoes, fruit and most dairy products. In PHASE 2, healthy carbs, including most fruits, whole grains and dairy products are gradually reintroduced, but processed carbs such as bagels, cookies, cornflakes, regular pasta and rice cakes remain on the list of foods to avoid or eat rarely.

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