Amplification by nucleic acid-templated reactions

Abstract

Nucleic acid-templated reactions enable the design of conditional reaction systems, in which bond formation occurs only when a particular DNA or RNA molecule is present. Such reaction systems are currently being explored for applications in DNA/RNA diagnosis, drug screening and as a means to design gene expression specific therapy. However, biological nucleic acid templates usually have low abundance. Therefore, either the targeted nucleic acid template has to be multiplied by means of an amplification step or the template itself has to act as a catalyst which amplifies product formation. This critical review highlights the recent advancements in nucleic acid-templated reactions that proceed with turnover in template and thereby provide a means of amplification. Improvements in reaction engineering and the development of new chemistries have pushed the limits from 10(1) to 10(2)-10(3) turnovers. This includes reaction systems that lead to the ligation of oligonucleotides or to the interconversion of appended functional groups beyond ligation as well as templated chemistries that enable the activation of catalysts for subsequent triggering of reactions between non-nucleotidic substrates. The present limitations and future opportunities are discussed.

Templated chemistry for bioorganic synthesis and chemical biology

“…PCR is not an option for reactions that ought to proceed in biological samples. Ideally, the templated reaction should provide for turnover in the target strand . In this case, each target molecule would instruct the formation of many product molecules.…”

“…Ideally, the templated reaction should provide for turnover in the target strand. 97,98 In this case, each target molecule would instruct the formation of many product molecules. However, nucleic acid-templated ligation reactions suffer from product inhibition.…”

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester

“…Various chemical ligation reactions have been applied to non-enzymatic sequencing of ONs (5), ON-based diagnosis (6), biotechnology (2,7) and nanotechnology for making functional nanoconstructs (8–11). In general, the chemical ligation between two functional groups requires the presence of condensing reagents.…”

“…Additionally, a variety of functional group pairs, for example, a nucleophilic group and an electrophilic group, or alkyne and azide groups, have been examined for use in chemical ligations with various applications in biology and biotechnology (2,20). ON-templated reactions without using any extra reagents were developed (2,7,21,22), and they are related to the study on the origin of life (23). These types of reactions are especially suitable for chemical ligations in biological contexts such as in cells, for RNA detection (3) or intracellular build-up of siRNA species (4).…”

Achieving room temperature DNA amplification by dialling in destabilization

“…[3][4][5] One major challenge facing nucleic-acid based point-of-care (POC) diagnostics is the low concentration of nucleic acid biomarkers in all biologically extracted samples. [6][7][8] As such, multiple examples of POC diagnostics using enzyme-based isothermal amplification methods have been reported that avoid the expensive instrumentation necessitated by the temperature cycling steps of the polymerase chain reaction. [6][7][8] As such, multiple examples of POC diagnostics using enzyme-based isothermal amplification methods have been reported that avoid the expensive instrumentation necessitated by the temperature cycling steps of the polymerase chain reaction.…”