Enzymatic Synthesis of Periodic DNA Nanoribbons for Intracellular pH Sensing and Gene Silencing

Abstract

We report the construction of periodic DNA nanoribbons (DNRs) by a modified DNA origami method. Unlike the conventional DNA origami, the DNR scaffold is a long, single-stranded DNA of tandem repeats, originating from the rolling circular amplification (RCA). Consequently, the number of folding staple strands tremendously decreases from hundreds to a few, which makes the DNR production scalable and cost-effective, thus potentially removing the barrier for practical applications of DNA nanostructures. Moreover, the co-replicational synthesis of scaffold and staple strands by RCA-based enzymatic reactions allows the generation of DNRs in one pot, further reducing the cost. Due to their unique periodicity, rigidity, and high aspect ratio, DNRs are efficiently internalized into cells and escape from endosomal entrapment, making them potential nanocarriers for imaging agents and biological therapeutics. We demonstrated proof-of-concept applications of DNRs as an intracellular pH sensor and an efficient small interfering RNA delivery vehicle in human cancer cells.

Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

“…The padlock probe and RCA method for target RNA detection is based on RNA-templated ligation. This ligation process is traditionally performed with T4 DNA ligase, 27,28 and more recently T4 RNA ligase 2, 29 and has been used for both mRNA 14,22 and miRNA detection. 29,30 However, the T4 enzymes are short of catalyzing ligation in the RNA template, thus reverse transcription was adapted for transforming the target RNA to cDNA to circumvent the limitation.…”

Self‐Assembled DNA Nanostructures for Drug Delivery

“…[71][72][73] RCAgenerated periodic DNA nanoribbons (DNRs) are able to work as intracellular pH sensors and efficient small interfering RNA delivery carriers in tumor cells. [74] RCA-based DNA nanostructures provide more choices and enable other programmed drug delivery platforms.…”

Rolling Circle Replication for Engineering Drug Delivery Carriers

“…For instance, Hamblin et al designed a DNA 'ladder' with RCA generated approximately 1400-15000 bp long ssDNA as the 'strut' and some other DNA oligos as 'rungs', resulting in very high aspect ratio of the final nanotube assembly [12]. Another version of RCR-based nanoassembly is the modified DNA 'origami', in which the generally used virus genomic DNA was replaced with RCR-generated DNA [13] or RNA [14] as the scaffold. In this strategy, the staples required to fold the scaffold were reduced from hundreds to only a few, which significantly simplified the folding process.…”

“…In this strategy, the staples required to fold the scaffold were reduced from hundreds to only a few, which significantly simplified the folding process. In the proof of principle study by Weizmann and coworkers [13], intact and digested RCR products were both used to synthesize scaffold and staples, respectively. High aspect ratio and rigidity of the folded nanoribbon enabled its efficient cellular uptake as well as endosome escape, making the nanoribbon a multifunctional platform for delivering various types of cargos, such as fluorescent dyes, protein and siRNA.…”