RNA and DNA molecules can form complex, three-dimensional folded structures that have surprisingly sophisticated functions, including catalysing chemical reactions and controlling gene expression. Although natural nucleic acids make occasional use of these advanced functions, the true potential for sophisticated function by these biological polymers is far greater. An important challenge for biochemists is to take RNA and DNA beyond their proven use as polymers that form double-helical structures. Molecular engineers are beginning to harness the power of nucleic acids that form more complex three-dimensional structures, and apply them as tools for exploring biological systems and as therapeutics.
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At a glance
: Manipulating the expression and function of proteins with nucleic acids.
Depicted is a simplified schematic of the flow of biological information from DNA to proteins and the points of intervention in this process by nucleic acid tools. Current or emerging technologies include: oligonucleotide- or vector-mediated genetic engineering
(1); triplex-forming oligonucleotides (TFOs) 109, 110 (2); ribozyme alteration of DNA sequence 111 (3); antisense oligonucleotides 112 (4); siRNAs 15, 16 (5); mRNA cleavage by ribozymes and deoxyribozymes 7 (6); ribozyme repair of mRNAs 35 (7); engineered riboswitches 112, 113 (8); aptamers 98, 99, 100, 101, 102, 103 (9); nucleoside-analogue drugs (10). Potential future technologies include: replacement of protein with functional DNA (11); repair of DNA by functional RNA or DNA (12); replacement of protein with functional RNA (13); modification of protein by functional RNA or DNA (14). 22, 23, 24, 26
: The generation and application of aptamers.
Methods for the
in vitro evolution of aptamers include the use of an affinity matrix to separate RNA variants that selectively bind an immobilized target (X; X represents any target molecule unless otherwise noted) , or the use of allosteric ribozymes that permit separation of ligand-binding RNAs by means of self-cleavage 22, 23, 26 . Once they have been engineered, aptamers can be used (among other applications) as chromatographic agents, biosensor elements, anti-protein drugs, gene-control elements and as components of allosteric ribozymes. 81, 82, 83
: RNA-cleaving ribozymes and deoxyribozymes.
The natural hammerhead ribozyme as well as the engineered X-motif ribozyme and 10–23 deoxyribozyme motifs catalyse RNA cleavage by promoting an internal phosphoester transfer reaction (inset). Base pairing between the RNA target and the substrate-binding arms of each catalyst can be tailored to target different RNA sequences. Nucleotides within the target RNA, the ribozymes and the deoxyribozyme that are not conserved are depicted with black, red and blue lines, respectively. B, base.
: Allosteric ribozymes as precision biosensor elements.
a, One of the first engineered allosteric ribozymes was created by fusing an ATP-binding aptamer to a hammerhead ribozyme by means of a disordered bridge element . Ligand binding stabilizes the core of the aptamer and the weakly pairing stem (stem II of the ribozyme) to trigger increased ribozyme activity. 80 b, A next-generation allosteric ribozyme or RiboReporter that senses ADP and disfavours binding of ATP by more than 100-fold. c, In the design shown here, a fluorescent readout is generated if ribozyme activity is triggered by ADP. As a result, fluorescence increase is prevented if an anti-protein-kinase drug, such as staurosoprine, is present . RNA cleavage by the ribozyme occurs within the stem I/III junction between A and G (blue arrow). F and Q represent fluorophore and quencher moieties, respectively. The performance characteristics of this RNA switch are sufficient to permit its use in high-throughput screening assays. 87
: Natural and engineered riboswitches for controlling gene expression.
a, A natural adenine-binding aptamer and its role in activating gene expression as part of an adenine riboswitch from the ydhL gene of Bacillus subtilis. The consensus sequence and secondary structure for the adenine aptamer domain is shaded. When sufficient adenine is present (top), the 5′ untranslated region (UTR) folds to form the full secondary structure for the aptamer bound to its ligand. This precludes nucleotides from forming an intrinsic terminator stem (shown in green) and a complete mRNA is synthesized. In the absence of adenine (bottom), portions of the secondary structure required for the aptamer to bind its ligand (red and blue nucleotides) are not formed, which permits the intrinsic terminator to form and cause premature transcription termination. Gene expression is prevented because the complete mRNA is not synthesized. 114, 115 b, Proposed mechanism for an engineered genetic switch that uses a theophylline-specific aptamer . The aptamer (shaded) and a short linker region is integrated with a 5′ UTR and the construct is fused upstream of an open reading frame. In the absence of theophylline (left), the protein is expressed because the RBS is available for interaction with ribosomes. In the presence of theophylline (right), the aptamer/linker structure becomes stabilized. This more stable structure presumably restricts ribosome access to the RBS, thus reducing gene expression. 103