Sequence Design
To make the GI-Nc using only DNA, the nanoclew template, the GI, the GcI, and the IcG sequences were designed to form the delivery vehicle to execute pH-dependent chemotherapy drug release and to bind photosensitizers for photodynamic therapy.
Nanoclew Synthesis
Duplex Formation
Design Process
Nanoclew Template
The nanoclew template has three main features:
- Palindromic sequence: The palindromic sequence is incorporated twice into the template and facilitates the folding of the amplified strand into the nanoclew structure.
- Overhang binding site: The overhang binding site is incorporated into the middle of the template and enables the attachment of drug-loaded duplexes and aptamers.
- Primer annealing site: The primer annealing site is composed of a region on each end of the template. These two regions are complementary to a single primer, thus circularizing the template when it binds.
Image 1: Linear (top) and circular (bottom) nanoclew template with primer binding sites in green, palindromic sites in orange, and the overhang binding site in yellow
The palindromic sequence, primer annealing site, and primer were based on the work of Ruan et. al [1]. The palindromic sequence and the primers were identical to their nanoclew design to match their process of assembly. Meanwhile, the overhang binding site is identical to the overhang we designed for the GI, GcI, and IcG. Therefore, when repetitively amplified to generate a long complementary strand, the nanoclew will contain multiple copies of a binding site complementary to the overhang to allow for the GI, GcI, and IcG to be attached.
GI, GcI, IcG
Image 2: Conformations of the GI (left), GcI (middle), and IcG (right) as determined by NUPACK
These strands encode for the following:
- GI: Both G-quadruplex and i-motif
- GcI: Only the G-quadruplex flanked by sequences without i-motifs
- IcG: Only the i-motif flanking a region of no G-quadruplex formation
The G-quadruplex binds zinc phthalocyanine for photodynamic therapy, and the i-motif enables the pH-dependent release of doxorubicin. Although the GI sequence is the one used in the final product, the GcI and IcG sequences were required to act as controls in independently testing the functionality of each component.
The GI and GcI were designed by Park et. al [2]. The IcG was designed by replacing the region encoding for the G-quadruplex with a string of thymines to eliminate quadruplex formation from the originally guanine-rich region. All three strands required the addition of an overhang to bind to the nanoclew, which was done by randomly generating DNA sequences that were 20 nucleotides in length. Eighteen nucleotides were used for annealing to the nanoclew and two were for reducing steric hindrance. NUPACK was used to find an overhang sequence that did not alter the intra-strand folding of the GI, GcI, and IcG at physiological temperature, so the formation of the i-motif and G-quadruplex would not be disrupted.
The GI, GcI, and IcG sequences are as follows:
GI: 5’ CCCCTTCCCCTGGGTGGGTTGGGTGGGTCCCCTTCCCC-3’
GcI: 5’ TTGGTGTTTTTGGGTGGGTTGGGTGGGTTTTTGTGGTT-3’
IcG: 5’ CCCCTTCCCCTTTTTTTTTTTTTTTTTTCCCCTTCCCC-3’
References
1. Ruan, W., Zheng, M., An, Y., Liu, Y., Lovejoy, D.B., Hao, M., … Shi, B. (2018). DNA nanoclew templated spherical nucleic acids for siRNA delivery. Chem Commun (Camb), 54(29), 3609-3612.
2. Park, H., Kim, J., Jung, S., & Kim, W.J. (2017). DNA-Au Nanomachine Equipped with i-motif and G-Quadruplex for Triple Combinatorial Anti-Tumor Therapy. Advanced Functional Materials, 28(5).
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