Nucleic acid backbone modifications

Both mechanical means and transfection reagents, among others, have been used to facilitate the cellular uptake of oligonucleotides. The application of intraluminal pressure enhances the uptake of particular oligonucleotides in vascular tissues such as carotid arteries or venous bypass grafts [14, 15]. Other approaches use chemical modifications in order to secondarily modify the nucleic acid backbone [16, 17]. In general, these modifications increase uptake through the cell membrane based on the classical receptor-mediated endocytosis pathway. However, once inside the cell, most nucleic acid compounds taken up by endocytosis are ultimately trapped in the lysosomal compartment... 

Figure 18.6. Covalent modification of CNTs by addition of ammonium ions promotes electrostatic interactions with the anionic nucleic acid backbone. ...

Fig. 1 Chemical structures of backbone modifications used in therapeutic nucleic acid analogs. Shown are the unmodified DNA/RNA chemical structures in addition to a selection of first (PS), second (OMe, MOE), and third generation (PNA, LNA, MF) nucleic acid modifications...

RNAi technology has obvious therapeutic potential as an antisense agent, and initial therapeutic targets of RNAi include viral infection, neurological diseases and cancer therapy. The synthesis of dsRNA displaying the desired nucleotide sequence is straightforward. However, as in the case of additional nucleic-acid-based therapeutic approaches, major technical hurdles remain to be overcome before RNAi becomes a therapeutic reality. Naked unmodified siRNAs for example display a serum half-life of less than 1 min, due to serum nuclease degradation. Approaches to improve the RNAi pharmacokinetic profile include chemical modification of the nucleotide backbone, to render it nuclease resistant, and the use of viral or non-viral vectors, to achieve safe product delivery to cells. As such, the jury remains out in terms of the development and approval of RNAi-based medicines, in the short to medium term at least. 

Any technique designed to characterize binding interactions by determining the accessibility of the backbone of macromolecules to cleavage or modification reactions. For nucleic acid interactions, footprinting was originally accomplished by changes in phosphodiester accessibility to DNase 1, but numerous chemical and enzymatic methods continue to be elaborated. 

Abstract Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific dehvery is also briefly... 

The research performed by several groups on pullulans has demonstrated their potential as nucleic acid delivery vehicles. Although most of the pullulan-based delivery systems yielded low toxicity, some modifications of the backbone or introduction of substituents resulted in higher toxicity. Such modifications are unavoidable because the parent structure is incapable of efficient delivery and lacks target specificity. 

Most synthetic DNA analogs represent logical departures from the natural structure. However, one of the most radical structural modifications of DNA to appear in the literature is peptide nucleic acid (PNA, Fig. 1), in which the sugar-phosphate backbone is abolished entirely in favor of a pseudopeptide. PNA was first reported by Nielsen et al. in 1991... 

Pokorski JK, Witschi MA, Purnell BL, Appefla DH. (S,S)-trans-Cyclopentane-constrained pephde nucleic acids. A general backbone modification that improves binding affinity and sequence specificity. J. Am. Chem. Soc. 2004 126 15067-15073. Govindaraju T, Kumar VA, Ganesh KN. (NR/RYj-Cyclohexanyl PNAs conformationally preorganized PNA analogues with unprecedented preference for duplex formation with RNA. J. Am. Chem. Soc. 2005 127 4144 145. 

Another approach being used to enhance the ionization process is the chemical modification of the nucleic acid molecule in order to improve ionic volatility. For example, the replacement of phosphate protons from native DNA backbones by alkyl groups,25 or the replacement of phosphate groups by phosphorothioate groups followed by alkylation26 have been reported. 

The reactivities of other transition-metal reagents have also been used advantageously in probing nucleic-acid stractures. As described in Section II, OSO4 reacts across the 5,6 position of accessible pyrimidines to form a cw-osmate ester. Upon treatment with piperidine, this base modification can yield scission of the sugar-phosphate backbone. Hence DNAs containing unusual local con-... 

Figure 8.7 Modification of oligonucleotides to increase stability, (a) Oligonucleotides (here shown as DNA) with a phosphodiester backbone (X = 0) are rapidly degraded by nucleases. Modification to create phosphorothioate analogs (X = S ) greatly increases half-life, (b) Peptide nucleic acids represent another DNA analog that can be used to bind with complementary sequences of oligonucleotides. Dashed lines represent hydrogen bonding which follows Watson-Crick base pairs.

---