- The Logic GateDNAzymes are DNA molecules that have the ability to perform a chemical reaction, such as catalytic action.
- Y-DNAY-DNA is composed of three ssDNA that is complementary of each other.
- The Origami AmplifierDNA origami is the nanoscale folding of DNA to create arbitrary two and three dimensional shapes at the nanoscale.
DNAzymes (also known as deoxyribozymes, DNA enzymes or catalytic DNA, are DNA molecules that have the ability to perform a chemical reaction, such as catalytic action. Since the description of the first DNAzyme for the cleavage of RNA in 1994, many more DNAzymes have been reported to catalyze many different types of chemical transformations, such as porphyrin metalation, DNA phosphorylation, RNA ligation, thymine-thymine dimer repair, carbon-carbon bond formation, and hydrolytic cleavage of DNA. DNA is chemically stable and can be conveniently produced by highly efficient automated DNA synthesis. Therefore, DNAzymes can be quite useful in research and applications in chemical biology, biotechnology, and medical areas.
In our project, we focus on the hydrolytic cleavage of DNA by DNAzyme. Cation-specific DNAzymes are composed of two functional domains: a catalytic loop that recognizes specific ions employed as coenzyme and a binding arm that targets its complementary sequence, or substrate. The sequence of the binding-arms can be altered at will so to adjust to different substrate. On the other hand, the catalytic activity of the DNAzyme relies on the catalytic core sequence which is conservative. The alteration of the conservative sequence will largely affect the catalytic activity. When the DNAzyme is hybridized with its target substrate sequence and the desired cation (cofactor) binds to the catalytic loop, hydrolysis of the target substrate sequences is activated. Because of the lower affinity of the cleaved substrate, the substrate will detach from the DNAzyme.
8-17 is the one of the most comprehensively studied RNA-cleaving DNAzymes and have the ability to carry out sequence-specific cleavage of all-RNA or chimeric RNA/DNA substrate (Figure 1.) 8-17 is small in size and composed of a catalytic domain flanked by two substrate binding arms. The catalytic domain of 8-17 has 15 nucleotides composed of a three basepair stem, an AGC triloop, and six unpaired nucleotides. One of the most significant features of 8-17 is the excellent selectivity. For 8-17, Pb2+ can activate it at very low concentration (<1μM) comparing with other ion. The substrate of 8-17 is RNA, at least nucleotide at the cut point should be ribonucleotide.
The Cu2+ DNAzyme is also an ssDNA that contains a stem-loop of 8 base-pairing. The catalytic domain consists of a conservative sequence of six basepair. The two binding arms flanking the catalytic domain bind with the substrate, one of which forms a DNA triplex of the stem-loop with the substrate. Unlike 8-17, the substrate of Cu2+ DNAzyme is deoxyribonucleotide. When the Cu2+ concentration is <1μM, DNAzyme is still activated. When other ions’ concentration is enormously bigger than Cu2+, the DNAzyme still didn’t recover its full activity, which shows its great selectivity of Cu2+.
There were examples of using DNAzyme to construct logic gate, but their input signals were ssDNA that which required multiple strand replacement. The reaction increases the response time. Due to this, we set out to design an AND gate that uses ion cofactors as input signals, because this mechanism reduced the reaction procedure. Furthermore, the excellent selectivity of metal ion of 8-17 and Cu2+ DNAzyme installs more advantage of our new design.
Y-DNA is composed of three ssDNA that is complementary of each other. Each of the branch can contain a sticky end. These ends can be linked with each other with the help of T4 ligase, and help Y-DNA to form a polymer. When and concentration and crosslinking degree is high enough, the polymer can turn into a hydrogel. The hydrogel can act as a drug delivery system, because of the biocompatibility. However, how much Y-DNA being produced and its decomposition cannot be easily controlled.
This year, we use our logic gate of 8-17 and Cu2+ DNAzyme to control the formation of Y-DNA. We optimized logic gate structure to suit the goal of controlling the formation and decomposition of Y-DNA.
DNA origami is the nanoscale folding of DNA to create arbitrary two and three dimensional shapes at the nanoscale. The specificity of the interactions between complementary base pairs make DNA a useful construction material, through design of its base sequences.
Developed by Paul Rothemund at the California Institute of Technology, the process involves the folding of a long single strand of viral DNA aided by multiple smaller "staple" strands. These shorter strands bind the longer in various places, resulting in various shapes, including a smiley face and a coarse map of China and the Americas, along with many three-dimensional structures such as cubes.
To produce a desired shape, images are drawn with a raster fill of a single long DNA molecule. This design is then fed into a computer program that calculates the placement of individual staple strands. Each staple binds to a specific region of the DNA template, and thus due to Watson-Crick base pairing, the necessary sequences of all staple strands are known and displayed. The DNA is mixed, then heated and cooled. As the DNA cools, the various staples pull the long strand into the desired shape. Designs are directly observable via several methods, including atomic force microscopy, or fluorescence microscopy when DNA is coupled to fluorescent materials.
This year, we used the design from 2012 Harvard BIOMOD team to build the origami. What’s different is that we load ssDNAs on the staple strand. The ssDNAs can serve as the substrate of logic gate of 8-17 and Cu2+ DNAzyme, thus the release can be controlled by it. This new origami can serve as a miRNA delivery system based on ion detection.