Abstract
Measuring at ~30 nm, a fully customizable holliday junction DNA nanoconstruct, was designed to simultaneously carry three unmodified SiRNA strands for apoptosis gene knockout in cancer cells without any assistance from commercial transfection kits. In brief, a holliday junction structure was intelligently designed to present one arm with a cell targeting aptamer (AS1411) while the remaining three arms to carry different SiRNA strands by means of DNA/RNA duplex for inducing apoptosis in cancer cells. By carrying the three SiRNA strands (AKT, MDM2 and Survivin) into triple negative breast MDA-MB-231 cancer cells, cell number had reduced by up to ~82% within 24 hours solely from one single administration of 32 picomoles. In the immunoblotting studies, up-elevation of phosphorylated p53 was observed for more than 8 hours while the three genes of interest were suppressed by nearly half by the 4-hour mark upon administration. Furthermore, we were able to demonstrate high cell selectivity of the nanoconstruct and did not exhibit usual morphological stress induced from liposomal-based transfection agents. To the best of the authors’ knowledge, this system represents the first of its kind in current literature utilizing a short and highly customizable holliday DNA junction to carry SiRNA for apoptosis studies.
Highlights
Aptamers, with its lower level of immunogenicity[27] as well as being more economically viable compared to antibodies, are often proposed as another alternative for cell targeting
We postulated that the cross-like contorted nano-architecture could offer much steric hindrance towards the ribonuclease antisense and this would help to reduce the overall processivity for the ribonuclease antisense to work on cleaving the DNA/RNA
It is important to note that if cleavage had occurred for the DNA/RNA duplex, calculations from closest neighbor parameter would shown that the fragmented RNA oligomers were incapable of forming stable duplex with mRNA at 37 °C
Summary
Aptamers, with its lower level of immunogenicity[27] as well as being more economically viable compared to antibodies, are often proposed as another alternative for cell targeting. DNA aptamer are short strands of DNA that can readily self-hybridized with itself to present important tertiary structures They can serve to bind to cell surface receptors and gaining entry into cell targets with high specificity[28,29,30,31]. Much of the current research has reported the use of aptamers to deliver single antisense RNA (double stranded) and most often involved covalent conjugation of the ends of the DNA aptamer directly to the functional end of the RNA or as “chimeras”[10, 38] These bioconjugated DNA/RNA nanocomplexes were administered directly to the designated cell, and gene suppression was subsequently measured. We were able to show that this nanoconstruct was able to induce high cancer cell apoptosis within 24 hours and could be tailored to suppress any gene of interest by the mere change of the sequence selection without relying on overly complicated bioconjugation processes
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