DNAzymes and Ribozymes - What are they?
Ribozymes are catalytic RNA molecules that break and form chemical bonds, proving that RNA can function as both genetic material and an enzyme. Thomas Cech discovered the first ribozyme in 1982 while studying self-splicing introns in the ciliate Tetrahymena thermophila. Sidney Altman discovered the second ribozyme, RNase P, showing that an RNA component catalyzes tRNA maturation. Nobel Prize 1989: Cech and Altman jointly received the 1989 Nobel Prize in Chemistry for their groundbreaking discovery.
This discovery shattered the dogma that biological catalysts had to be proteins and laid the foundation for the RNA World hypothesis, which suggests early life relied on RNA for both information storage and catalytic activity.
DNAzymes differ from Ribozymes
A DNAzyme, or deoxyribozyme, is a catalytic nucleic acid made of DNA, whereas a ribozyme is a catalytic nucleic acid made of RNA. DNAzymes consist of single-stranded deoxyribonucleotides that lack a 2'-hydroxyl group, while ribozymes consist of ribonucleotides that have a 2'-hydroxyl group.
Ribozymes occur widely in nature, including hammerhead ribozymes and the ribosome, and support the RNA World hypothesis. DNAzymes are almost entirely artificial, created through in vitro selection via laboratory evolution, with little to no evidence of natural counterparts.
Ribozymes were discovered in the early 1980s, revolutionizing the dogma that only proteins act as enzymes. Breaker and Joyce developed the first DNAzyme in 1994.
DNAzymes are generally more chemically stable and less expensive to manufacture than ribozymes because DNA is inherently more resistant to spontaneous cleavage and hydrolysis. Both DNAzymes and ribozymes frequently require metal-ion cofactors, such as Mg²⁺, Zn²⁺, or Pb²⁺, to carry out catalysis. DNAzymes are widely used in metal-ion sensing and biosensors because of their strict cation-dependent specificity.
In target-cleavage applications, DNAzymes often favor slightly longer recognition or binding arms than ribozymes to achieve high catalytic efficiency.
Several 3D structures have been solved for DNAzymes and ribosome. Below are two examples.
| 8-17 DNAzyme: 8OR8: Solution structure of the 8-17 DNAzyme in presence of Zn2+ | 1HMH: THREE-DIMENSIONAL STRUCTURE OF A HAMMERHEAD RIBOZYME |
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| Wieruszewska et al. (2024) reported the solution structure of 8–17 DNAzyme bound to a Zn2+ ion. The authors use solution-state NMR to characterize the structure of the 8–17 DNAzyme, revealing that all metal cofactors induce the same DNAzyme fold in contrast to previous findings. This NMR investigations in the presence of other ions — Mg2+, Na+, and Pb2+ – suggested that at DNAzyme concentrations used in NMR all these ions induce a similar tertiary fold. | Plesy et al. (1995) solved the 3D structure of a hammerhead ribozyme, small catalytic RNA motif made up of three base-paired stems and a core of highly conserved, non-complementary nucleotides essential for catalysis. The base-paired stems are A-form helices and the core has two structural domains. The first domain is formed by the sequence 5'-CUGA following stem I and is a sharp turn identical to the uridine turn of transfer RNA. The second is a non-Watson-Crick three-base-pair duplex with a divalent-ion binding site. The phosphodiester backbone of the DNA inhibitor strand is splayed out at the phosphate 5' to the cleavage site. The ribozyme may destabilize a substrate strand in order to facilitate twisting of the substrate to allow cleavage of the scissile bond. |
Synthesis of DNAzymes
Because no natural DNAzymes exist, researchers synthesize and discover them primarily through in vitro selection (SELEX) or build them via solid-phase DNA synthesis once a catalytic sequence is known. Rather than rationally designing DNAzymes from scratch, researchers evolve them through iterative synthesis cycles.
For library construction, researchers chemically synthesize a large combinatorial library of single-stranded DNA using solid-phase synthesis methods. Each strand contains a central region of random nucleotides, typically 25 to 50+ bases long, flanked by constant primer-binding sites needed for subsequent polymerase chain reaction (PCR) amplification, creating a sequence space of up to 4N unique variants, in the range of 10¹⁴ to 10¹⁵ individual molecules.
Typically, during the incubation step, a DNA pool is exposed to a target substrate, such as an RNA-containing strand, with cofactors, commonly divalent metal ions such as Mg2+ or Pb2+.
Active DNA strands that catalyze the desired reaction, for example, cleaving an RNA bond or self-phosphorylating, are physically separated from inactive sequences. Common separation methods include affinity chromatography or gel electrophoresis. The isolated active DNA sequences are amplified by PCR to generate an enriched pool for the next selection round. Repeat the selection process for 5 to 10+ rounds with increasing stringency until highly efficient catalytic sequences dominate the pool.
Synthesis of Ribozymes
Ribozymes can be synthesized and generated through three main methods:
[1] in vitro transcription,
[2] chemical synthesis, and
[3] in vitro evolution/selection.
In Vitro Transcription
RNA polymerases, such as T7 RNA polymerase, use a DNA template to synthesize ribozymes enzymatically in a test tube. This is the most common method for producing functional, full-length ribozymes like group I introns, hammerhead ribozymes, or RNA polymerase ribozymes.
Chemical Synthesis
Automated solid-phase chemical synthesis builds RNA chains nucleotide by nucleotide, allowing researchers to stitch together shorter synthetic RNA fragments (36, 43, or 59 nucleotides) that can self-assemble into a catalytically active complex, or to incorporate modified ribonucleotide analogs site-specifically.
In Vitro Evolution & SELEX
Systematic Evolution of Ligands by Exponential Enrichment (SELEX) enables the creation and improvement of new ribozymes. Screening large libraries of random RNA sequences can detect and identify novel RNA sequences with catalytic activity.
Template-Directed Polymerization
Advanced synthetic ribozymes, such as RNA polymerase ribozymes, can catalyze the synthesis of new RNA strands from activated monomers or trinucleotide triphosphate triplets.
Metal-Ion Catalysis
Most ribozyme synthesis and cleavage reactions require divalent cations such as magnesium (Mg2+) to stabilize secondary and tertiary RNA folding and to facilitate acid-base chemistry.
Self-Assembly
Certain engineered ribozymes are designed to synthesize their own constituent subunits in segments ("+" and "–" strands), which then spontaneously fold and assemble into active ribozyme complexes.
References
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Deoxyribozyme - an overview
Deoxyribozyme
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Ribozyme
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