Surface-bound DNA self-assembly or enzymatic reactions toward the PCR-free amplification of nucleic acids biosensor signals

INTRODUCTION: Single-molecule sensitivity can be nowadays achieved when trying to characterize minute amounts of macromolecules n in a research laboratory setting. The sensitivity is significantly worse when working in the field and trying to assess the presence of a pollutant or a pathogen as quickly as possible. Point-of-care testing is the realm of biosensors and the trend of research is towards simplicity of use, low cost, reliability and, last but not least, sensitivity. In the detection of nucleic acids, the hybridization of an oligonucleotide probe with its poly-nucleotidic target is monitored by changes in physico-chemical properties of an interface, commonly induced by the use of a specific label. The probe, often bound to an oligonucleotide itself, can be a fluorescent dye, an enzyme, an electroactive moiety or other functional element. Such probes impart specificity and sensitivity to the assay, but make it drift far from simplicity of use (and low cost). METHODS AND RESULTS: Recently, label-free techniques are developing in which the presence of the analyte macromolecule itself can induce detectable physico-chemical changes. The struggle is to make such techniques sufficiently sensitive and specific. In our work, we are trying to implement DNA-based surface-bound amplification strategies that can serve to amplify the read-out signal of DNA hybridization in a label-free biosensor, such as those measuring the electrochemical properties of an electrode interface. The ‘hybridization chain reaction’ [1] is an isothermal enzyme-free strategy to trigger polymerization of oligonucleotides into a long double-stranded DNA. We have demonstrated that the ‘hybridization chain reaction’ can be also implemented (making use of available software tools [2]) in a surface-bound configuration, leading to the self-assembly of many copies of oligonucleotides on a target DNA bound on an oligonucleotide self-assembled monolayer. In another attempt, we have shown that terminal transferase (a template-free DNA polymerase) can be used to build a long polynucleotide out of the target DNA that is bound to its immobilized oligonucleotide probe. The Rolling Circle Amplification reaction is a very promising strategy to produce large amounts of DNA without the need of thermal cycling or of an accurate control of temperature. Preliminary evaluations on the Primer-Generation RCA [3] show its interest as an additional strategy to increase the amount of target DNA response from a biosensor surface. DISCUSSION & CONCLUSIONS: All these strategies will lead to the accumulation of nucleic acids at the solid-liquid interface when triggered by the probe-target recognition. This produces an amplification of the signal of a label-free biosensor. Even though the amplification factors of these implementations are still as low as 10, their further development is still possible, with the hope of avoiding or reducing the need of polymerase chain reaction in the detection of low concentrations of nucleic acids. REFERENCES: 1Dirks, R. M. and N. A. Pierce (2004). "Triggered amplification by hybridization chain reaction." Proc Natl Acad Sci U S A, 101(43): 15275-8. 2Goodman, P. (2005). "NANEV: a program employing evolutionary methods for the design of nucleic acid nanostructures" BioTechniques, 38(4): 548–550. 3Murakami, Sumaoka and Komiyama (2009) "Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification" Nucleic Acids Research, 2009, 37, e19. ACKNOWLEDGEMENTS: This work was supported by Framework Programme 6 Integrated Project DINAMICS.