Nucleic acid dendrimers

The immobilization of the hyperbranched spherical structures onto physical transducers greatly increases the binding capacity of the surface and leads to enhanced sensitivity and extended linearity of biosensors. Nucleic acid dendrimers were prepared and their amplification properties for the detection of DNA were examined using mass-sensitive transducers [45, 46]. Antibodies... 

Electrophoresis is normally run in aqueous media, hence the analytes must be soluble in water. Presently only three types of water-soluble dendrimers have been successfully analyzed using gel electrophoresis techniques. The list includes Starburst PAMAM dendrimers [21], nucleic acid dendrimers [21] and poly(lysine) dendrimers [23, 24] (see Figures 10.2, 10.4 and 10.6). However, in each case appropriate water solubilizing terminal groups are required (i.e. -NH2, -OH or C02H groups) for suitable electrophoretic analysis. 

In another study, nucleic acid dendrimers were synthesized via both convergent and divergent approaches, and their purity assessed using PAGE [23, 24], In Figure 10.6 is shown convergent synthetic route, which involves ... 

In a recent report, the divergent solid-phase synthesis of nucleic acid dendrimers was also reported and the purity assessed using PAGE [24], however, the details are not included here due to space constraints. 

Figure 10.6 Synthetic scheme of nucleic acid dendrimers...

Access to nucleic acid dendrimers is initiated by a zip-fastener like dissociation of the DNA double strand by heating. The double strand separates into the two individual strands by thermal motion (denaturation). Subsequent association, hybridisation of complementary sequences, is followed by stepwise cross-linking to form DNA dendrimers, which can contain up to two million oligonucleotide-end group strands (Fig. 8.19). The latter can be labelled with fluorescence or radioactive markers. 

As non-viral delivery vectors of nucleic acids, dendrimers have shown to be able to form stable dendriplexes by electrostatic interaction with nucleic acids these dendriplexes in general are biocompatible over a wide concentrations range, protect the nucleic material from attack of serum proteins or nucleases and are able to internalize these nucleic acids in a large number of cell-lines and primary cell cultures with great efficiency. 

Hudson RHE, Damha MJ (1993) Nucleic-acid dendrimers—novel biopolymer structures. J Am Chem Soc 115 2119-2124... 

PAMAM dendrimers have the following characteristics which are important for their use as transfection reagents. They bind and form complexes with nucleic acids, allow transfer of the DNA-dendrimer complex into the cytoplasm of the... 

The latter is an interesting example of self-organizing chiral dendrimers. The construction of the dendrimer is based on the natural property of nucleic acids to recognize and specifically bind to complementary sequences. Pairwise hybridization of two designed DNA strands results in the formation of large monomers which have four single stranded arms and a double stranded waist (24, Fig. 12). 

The surface of each layer has two types of single stranded arms (e.g. one 3 — 5 and one 5 — 3 strand) which can bind to other monomers to render 1st-and 2nd-generation dendrimers 25 and 26. Therefore the molecular scaffold grows exponentially with each sequential layer of hybridization. If an oligonucleotide contains a sequence complementary to those at the surface of these networks it should be hybridized. The remaining free sequences from the other type of arms then bind in a standard nucleic acid blot (after they are bound to... 

Yoo, H., and Juliano, R.L. (2000) Enhanced delivery of antisense oligonucleotides with fluorophore-con-jugated PAMAM dendrimers. Nucleic Acids Res. 28, 4225-4231. 

Benters, R. Niemeyer, C. M. Wohrle, D., Dendrimer activated solid supports for nucleic acid and protein microarrays, Chembiochem. 2001, 2, 686 694... 

Gel electrophoresis is widely used in the routine analysis and separation of many well-known biopolymers such as proteins or nucleic acids. Little has been reported concerning the use of this methodology for the analysis of synthetic polymers, undoubtedly since in many cases these polymers are not soluble in aqueous solution - a medium normally used for electrophoresis. Even for those water-soluble synthetic polymers, the broad molecular weight dispersities usually associated with traditional polymers generally preclude the use of electrophoretic methods. Dendrimers, however, especially those constructed using semi-controlled or controlled structure synthesis (Chapters 8 and 9), possess narrow molecular weight distribution and those that are sufficiently water solubile, usually are ideal analytes for electrophoretic methods. More specifically, poly(amidoamine) (PAMAM) and related dendrimers have been proven amendable to gel electrophoresis, as will be discussed in this chapter. 

Structure controlled dendritic polymers that have been studied using gel electrophoresis generally behave as mimics of either proteins or nucleic acids, and possess similar ionic groups such as -NH3 , -COO , or P04 functionality. Dendrimer structures may be widely modified as a function of their interior composition and as well as the nature of their surface groups. Depending on their structure, the influence of pH may vary dramatically for different dendrimers. 

In summary, dendrimers are a unique class of monodispersed synthetic molecules reminiscent of proteins or nucleic acids. If they can be functionalized to be soluble in water with appropriately charged terminal groups, they are generally ideal candidates for gel electrophoretic analyses. 

Physical or electrochemical adsorption uses non-covalent forces to affix the nucleic acid to the solid support and represents a relatively simple mechanism for attachment that is easy to automate. Adsorption was favoured and described in some chapters as suitable immobilization technique when multisite attachment of DNA is needed to exploit the intrinsic DNA oxidation signal in hybridization reactions. Dendrimers such as polyamidoamine with a high density of terminal amino groups have been reported to increase the surface coverage of physically adsorbed DNA to the surface. Furthermore, electrochemical adsorption is described as a useful immobihzation strategy for electrochemical genosensor fabrication. 

Figure 3.17 Creation of dendrimer spacers. (From Beier, M. and Hoheisel, J.D., Nucleic Acid Res., 27(9), 1970-1977, 1999. With permission.)...

Bielinska, A., Kukowska-Latallo, J.F., Johnson, J., Tomalia, D.A. and Baker, Jr., J.R. (1996) Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acids Res., 24, 2176-2182. 

---