ABSTRACT
Post-transcriptional modifications on the guide RNAs utilized in the Cas9 system may have the potential to impact the activity of Cas9. In this study, we synthesized a series of tracrRNAs containing N 6-methyadenosine (m6A), a prevalent post-transcriptional modification, at various positions. We evaluated the effect of these modifications on the DNA cleavage activity of Cas9. Our results show that multiple m6As in the anti-repeat region of tracrRNA reduce the DNA cleavage activity of Cas9. This suggests that the m6A-modified tracrRNA can be used for Cas9 only when the number and the position of the modified residue are properly chosen in tracrRNA.
ABSTRACT
Aptamer-drug conjugates (ApDCs) are promising anticancer therapeutics with cancer cell specificity. However, versatile in vivo applications of ApDCs are hampered by their limited serum stability and inability to reach the tumour upon systemic administration. Here, we describe DNA nanoparticles of ApDCs as a platform for tumour-targeted systemic delivery of ApDCs. DNA nanoparticles of approximately 75 nm size were fabricated by self-assembly of a polymerised floxuridine (FUdR)-incorporated AS1411 aptamer produced via rolling circle amplification. The DNA nanoparticles of ApDCs showed highly efficient cancer cell uptake, enhanced serum stability, and tumour-targeted accumulation. These properties could be successfully utilised for tumour-specific apoptotic damage by ApDCs, leading to significant suppression of tumour growth without considerable systemic toxicity. Molecular analysis revealed that the enhanced anticancer potency was due to the synergic effect induced by the simultaneous activation of p53 by AS1411 and the inhibition of thymidylate synthase by FUdR, respectively, both of which were generated from the DNA nanoparticles. We therefore expect that the DNA nanoparticles of ApDCs can be a promising platform for tumour-targeted delivery of various nucleoside-incorporated ApDCs to treat cancer.
