Enhanced Diagnostic Tools
Abstract— Guanidine-based peptide nucleic acid (GPNA) monomers and oligomers containing all four natural (adenine (A), cyto- sine (C), guanine (G), and thymine (T)) and two unnatural (2-thiouracil ( s U) and 2,6-diaminopurine (D)) nucleobases have been synthesized. Thermal denaturation study showed that GPNA oligomers containing alternate D-backbone configuration bind sequence-specifically to DNA and, when incubated with mammalian cells, localized specifically to the endoplasmic reticulum (ER).
Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA, developed more than a decade ago in which the naturally occurring sugar phosphate backbone has been replaced by the N-(2-aminoethyl) glycine units.1 PNA can hybridize to complementary DNA or RNA strand through Watson–Crick base-pairing;2 but unlike DNA or RNA which is susceptible to enzymatic degradation, PNA is not easily degraded by proteases or nucleases.3 These properties make PNA an attractive reagent for biotechnology applications.
While several PNA-based applications have been successfully developed,4–6 the full potential of PNA for in vivo applications has not yet been realized—much of which could be attributed to its poor cellular uptake property. Considerable effort has been vested in the last decade trying to develop means to transport PNA into cells. Several strategies,7,8 including microinjection,9 electroporation,10 DNA-mediated transduction,11 and covalent attachment to cell-penetrating peptides (CPPs),12,13 have been developed, but for the most part they are limited to small-scale experimental setups. The latter approach, although promising since no delivery system is required, is presently limited by uptake efficiency and intracellular distribution. Most PNAs delivered this way are trapped in the endocytic vesicles.7 In addition, since these PNA–CPP constructs are amphipathic in nature, they could cause cytotoxic effects since most amphipathic peptides found in nature, such as magainin and mellitin, are known to be cytotoxic.14
Recently, we have shown that arginine-derived, polythymine PNA oligomers (commonly known as GPNAs) were readily taken-up by mammalian cells.15 Herein, we report the synthesis of GPNA monomers for all four natural (A, C, G, and T) and two unnatural (sU and D) nucleobases and the corresponding oligomers, along with their hybridization and cellular uptake properties. The synthetic strategy reported herein has two distinct advantages over the conventional design, where the transduction domains are covalently linked either to the C- or N-terminus of PNA. First, it minimizes the amphipathic character of the resulting complex, thereby reducing its cytotoxic effects. And second, it eliminates the extra synthetic steps required for appending molecular transporters to the termini of PNAs. GPNAs containing alternate N-(2-aminoethyl) D-arginine and N-(2-aminoethyl) glycine backbone units bind sequence-specifically to DNA (and RNA),16 and are taken-up by mammalian cells and localized specifically to the endoplasmic reticulum (ER).
GPNA monomers were synthesized according to the procedure outlined in Scheme 1. A similar procedure has been reported previously,17,18 but this particular protocol employed dicarboxybenzyl (diCbz)-protection for the arginine side chain and was limited to thymine
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