Enhanced resistance to nuclease degradation of nucleic acids complexed to asialoglycoprotein-polylysine carriers

Abstract

We have previously shown targeting of DNA to hepatocytes using an aslaloorosomucold-polylysine (AsOR-PL) carrier system. The AsOR-PL conjugate condenses DNA and facilitates entry via specific receptor-llgand Interactions. In these studies, our objective was to determine If AsOR-PL conjugates protect bound DNA from nuclease attack. Double-stranded plasmld or single-stranded ollgonucleotlde DNA, alone or bound to conjugate, was Incubated under conditions mimicking those encountered during In vitro and In vivo transfectlons. The results showed that complexed DNA was effectively protected from degradation by serum nucleases. Degradation of single-stranded ollgonucleotldes was Inhibited 3- to 6-fold in serum during 5 hours of Incubation. For complexed plasmlds, greater than 90% remained full-length during 1.5 and 3 hour incubations in serum or culture medium containing 10% serum, respectively. Uncomplexed plasmld was completely degraded after 15 minutes In serum or 60 minutes In medium. In cell lysates, the conjugate was not effective in inhibiting endonuclease activity; plasmids were readily converted from supercolled to open circular and linear forms. However, the resultant nicked forms were substantially protected from further degradation during one hour of incubation compared to plasmid alone. Under all conditions complexed DNA did not readily dissociate from the conjugate. Overall, for both single and double-stranded DNA, AsOR-PL conjugates conferred substantial protection from nuclease degradation. © 1994 Oxford University Press.