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DNAzymes
- 2. Definition and basics In vitro selection Types of reactions catalyzed DNAzyme Delivery Applications DNA Nanomotors DNAzyme Biosensors
- 3. Catalytically active DNA molecules Single stranded DNA Novel approach, no natural DNAzymes found DNA is more stable than RNA Less expensive to synthesize by solid phase synthesis Easier to synthesize than protein One DNAzyme ,one product as expected for ‘enzymes’ First DNAzyme was reported Breaker and Joyce, 1994
- 4. A random pool of DNA is made by solid phase synthesis Incubation under suitable reaction conditons 50mM Tris, pH 7.5,divalent metal ions (MgCL2), monovalent metal ions (NaCl), temperature 37 ° C Incubation time can be in hours or in minutes Every sequence should be attached with the substrate Only a few sequences will successfully catalyze the substrate
- 5. Selection strategy is devised to physically separate catalytically active DNA sequences After isolating active sequences, PCR amplification is used Whole procedure is iterated 5-15 times Increasing selection pressure Cloning final sequences in vector for sequence studies In vitro evolution can also be done so that the experiment is started with biased pool
- 6. RNA Cleavage RNA Ligation Branched RNA formation Lariat RNA formation Thymine dimer photocleavage
- 7. 10-23 DNAzyme can cleave any RNA sequence at a cleavage site which has an unpaired purine (A or G) followed by a paired pyrimidine (U or C) in the presence of Mg+2 (5’-R ↓ Y-3’) 8-17 DNAzyme cleaves 5`-A ↓ G-3` Cleavage site should have other RNA nucleotides on both sides that must be Watson-Crick base-paired with DNAzyme binding arms Used to target beta-lactamase and thus inhibit bacterial growth.
- 8. Two possible reactions can happen either native 3’-5’ or non native 2’-5’ Selection strategy was designed to favor only native linkage by introducing 8-17 DNAzyme. It is highly selective for cleaving 3’-5’ linkage Mg+2 dependent 9DB1 DNAzyme requires D↓RA where D denotes any nucleotide except C Zn+2 dependent 7DE5 requires only A ↓R
- 9. 7S11 DNAzyme forms a 3 helix junction with two RNA substrates. This arrangement provides preorganization for the nucleophile (2`OH) and electrophile (5`ppp) via formation of four paired regions denoted P1-P4 DNAzyme-catalyzed labeling (DECAL) treats one RNA substrate as the ‘target’ RNA to be labeled, and the second RNA substrate as a ‘tag’ to be attached by a DNAzyme such as 10DM24 at a predefined 2’-position of the target. When the ‘tagging’ RNA has a biophysical label (e.g., fluorescein or biotin) present at its second nucleotide, the DECAL strategy effectively labels the target RNA at a specific site.
- 10. Lariat formation is catalyzed by 6BX22 DNAzyme. The two reacting groups 2’OH and 5’ppp are part of the same nucleotide leading to the closed loop Using this enzyme lariats that have as many as 266 nucleotides have been created with high selectivity
- 11. UV1C DNAzyme uses light of λmax 305nm to promote the photochemical reaction. A two-tired guanine -quadruplex within the DNAzyme acts as an ‘antenna’ for subsequent electron transfer to the thymine dimer substrate. No cofactors are required
- 12. With the aid of the ‘‘sticky ends’’ of DNA, dispersed three- and four-way DNA junctions and other structural blocks can be connected into large repeating structures, on which nanoparticles or proteins can be deposited Atelocollagen, chitosan nanoparticles, and polypropylene imine have serve as alternative approaches to liposomes All these approaches have been used to deliver siRNA sequences, still not used for DNAzymes
- 14. Composed of two oligonucleotide strands. One strand (blue) contains 10-23,and the other strand (cyan) contains a donor-acceptor pair of fluorophores, F1 and F2, on opposing ends. The presence of the two fluorophores allows any change in motion to be observed through fluorescence resonance energy transfer (FRET). The motor is fueled by a chimeric (DNA/RNA) oligonucleotide substrate (red), which is susceptible to cleavage by 10-23. In the absence of the substrate, the motor adopts a ‘‘closed’’ conformation, characterized by a low fluorescence signal. However, when the substrate hybridizes to the DNAzyme, the DNA motor adopts an ‘‘open’’ conformation that leads to an increased fluorescence signal. Cleavage and subsequent dissociation of the substrate allows the DNA motor to once again adopt the closed conformation. This motor continues to cycle between the open and closed states as long as substrate is available.
- 15. A Pb2+ biosensor was constructed with a fluorescent reporting system, by simply labeling the 5’ end of the substrate oligonucleotide with a fluorophore, and the 3’ end of the 8-17 DNAzyme strand with a fluorescence quencher. In the uncleaved state, the substrate binds to the DNAzyme, positioning the fluorophore and quencher in proximity to each other for maximal fluorescence quenching. When Pb2+ is introduced into the solution, the DNAzyme becomes active and cleaves the substrate, which subsequently dissociates from the DNAzyme to generate a fluorescence signal.
Editor's Notes
- Good Morning everyone. I am here to speak about DNAzymes.
- I will cover the following topics.
- We always think DNA as two antiparallel strands arranged in a double-helical structure. However, DNA can be readily synthesized as a single-stranded polymer that can adopt many other types of structures, which is an indication that it can act as catalysts. These catalytic DNA molecules are commonly referred to as DNAzymes, or deoxyribozymes. We have heard about ribozymes. But DNAzymes are synthetic. Now, as we have proteins, ribozymes why do we need to create DNAzymes ? First because DNA phosphodiester bonds are 1000-fold more resistant to hydrolytic degradation than are peptide bonds. And also absence of a 2’-OH group at each phosphodiester linkage makes DNA 100,000-fold more stable to hydrolysis than RNA under physiological conditions. Solid phase synthesis is a technique used to synthesize oligopeptides, oligonucleotides, oligosachharides. Oligonucleotides are synthesized by THE PHOSPHORAMIDITE METHOD, here a nucleotide is added one by one in 3` to 5` direction. It is easier to synthesize because there are only 4 nucleobases A,T,G,C as compared to 20 amino acids in proteins. They cut RNA target at only one specific phosphodiester linkage, even though all other every other linkage has 2` OH. First was reported in 1994. It was a 38 nt ssDNA molecule, which catalyzed the Pb2+-dependent cleavage of an RNA phosphoester embedded within a separate DNA molecule.
- As I said they are not naturally found, they are created. So what we do is, we create a random pool of DNA sequences, feed them the substrate, incubate them, and then select the sequences that acted upon our substrate. In vitro selection is the standard procedure for it. Solid phase synthesis is used to create a random pool of all possible DNA sequences. The concentrations of phosphoramidites in the mixture are adjusted acc to the known relative reactivities, T>G>C>A. So that each of the four monomers has an equal chance. Now by random I mean there is a central sequence whose length is defined but sequence is random. This means if we decide to take a 40 length sequence then 440 ~ 1024 sequences are possible. For practical reasons a selection experiment can begin with only 1014 molecules. So the other sequences are not considered. We can also choose a length higher or lower than 40. Successful results have come out with both higher or lower lengths. So this would we a judgment call. Also, the random portion is flanked by fixed regions, required for PCR step. See the incubation conditions can have a wide range. If you are getting what I am saying then you can understand that there are lot of possibilities in this kind of experiment and you need to be lucky to get result.
- Strategy can be PAGE. For eg. for RNA ligation reactions, active sequences will be longer in length. Also for RNA clevage reactions, they will be shorter. Biotin-Streptavidin strategy can also be applied. Again a judgement call. Incubation conditions and time can be changed in order to get more efficient DNA sequences. This is like survival of the fittest theory. Biased pool can be made by altering the phosphoramidites conc. Or by using mutagenic PCR.
- There are several reactions that can be catalyzed by DNAzymes. We are going to cover the following.
- First isolated DNAzyme was ‘10–23’ DNAzyme’ described by Santoro and Joyce in 1997 & 1998. It was derived from the 23rd clone from the 10th round of PCR. Similarly ‘8-17’ was derived from the 17th clone from round 8. The 10–23 DNAzyme is composed of a catalytic domain of 15 deoxyribonucleotides flanked by two substrate recognition arms. Various variants of these two DNAzymes have been prepared and used in the industry. Three optimal 8-17 variants can cleave 10 out of 16 possible RNA junctions.
- After PAGE step in selection step, 8-17 is introduced which cleaves native bonds now again PAGE is done to get desired sequences.
- Lariat RNAs are a subclass of 2’,5’-branched RNAs in which two of the oligonucleotide arms emerging from the branch site are connected to form a closed loop. The branched RNAs formed during biological splicing are actually lariat RNAs.