Molecular Diagnostics; Overcoming target inhibition in nucleic acid-templated ligation reactions

The genetic information stored in DNA and RNA, dictates the production of proteins, the machinery of life. When mutations occur in these vital nucleic acids, proteins having an altered functioning can be formed disrupting normal cellular processes. Genetic disorders can be involved in severe illnesses such as Tay Sachs disease, Huntington’s disease, Alzheimer’s disease, Duchenne muscular dystrophy, cystic fibrosis, sickle cell anemia, Crohn’s disease and several types of cancers. An early diagnosis will allow a faster treatment and thus might improve patient recovery/survival, making specific disease related nucleic acids interesting diagnostic targets. A potentially useful property of nucleic acids is their specificity in forming duplexes. A single strand nucleic acid is able to hybridise to it’s complementary strand in a giant pool of nucleic acids, discriminating even a single-base mutant. Diverting from natures intended use of these information carriers, nucleic acids can also be used as efficient templates in organic reactions. The increase of effective molarity and proper alignment of probes upon hybridisation to a DNA template provides an environment for reactions to occur that under non-templated conditions would not. Combining the target specificity and potential as catalytic template, opens the way to an exciting scope of applications, such as the early stage detection of disease related nucleic acids. Detection of small amounts of nucleic acids however raises the need for a proper signal amplification pathway. Turnover numbers of nucleic acid templates are often limited. The aim in this proposal is to develop PNA-probes that in a DNA/RNA-templated fashion undergo ligation reactions in such a manner that a high-turnover number can be realised. The fluorescent signal formed by ligation of the PNA-probes will be amplified by the catalytic role of the template allowing a quick increase of fluorescent reaction products. This will enable the detection of disease related DNA at very low concentrations.