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Diagnostic applications of nucleic acid circuits.

ABSTRACT: CONSPECTUS: While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston-Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options.

SUBMITTER: Jung C

PROVIDER: S-EPMC4063332 | biostudies-other | 2014 Jun

REPOSITORIES: biostudies-other

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Project description:Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have potential therapeutic applications. They have extremely high thermodynamic stability, have increased resistance to enzymatic degradation, and have been investigated as decoys for regulatory proteins. For potential nanotechnology applications, we have synthesized double stranded DNA catenanes by click ligation. Other researchers have studied covalently fixed multistranded DNA constructs including triplexes and quadruplexes.

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Diagnostic applications of nucleic acid circuits.

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