Research database

Peptide nucleic acids (PNAs) are one of the best-performing tools in chemical biology for DNA and RNA recognition. PNA properties can be modulated by chemical modification to improve target affinity/selectivity and to introduce reporter or functional groups to create smart probes to develop ultra-sensitive detection techniques. To achieve these goals, fundamental research will be carried out, starting from the basic properties of PNAs and their interactions with microRNAs (miR), one of the most studied classes of cancer biomarkers. SEMPER aims to merge the state of the art from organic chemistry, chemical biology, and nanophysics to develop innovative dual probes, for fluorescence and Surface-Enhanced Raman Scattering (SERS) based assays to quantify miRs down to fM concentration. PNAs can form heteroduplexes with complementary DNA/RNA characterized by higher stability in comparison to the canonical duplexes, which can be translated into higher sensitivity towards short sequences of nucleic acids like miRs. PNAs’ higher robustness and straightforward preparation compared to DNA/RNA can better tolerate both the functionalization along the sequence and the presence of small functional molecules working as fluorescent and Raman reporter for SERS investigation. Two synthetic orthogonal yet complementary strategies will produce suitable PNAs able to provide emission and Raman signal without lowering their high affinity and selectivity towards the target miRs. The former aims to obtain the first complete emissive isomorphic genetic alphabet for PNAs, based on thieno[3,4-d]pyrimidine core, allowing the preparation of intrinsically fluorescent PNAs. The latter will focus on the functionalization of novel environmentally sensitive and intercalating naphthalenediimides (NDI) as PNA tags, able to work at the same time as fluorescence and Raman reporter. The intrinsically emissive PNAs and the NDI-tagged PNA will be employed to detect short miRs by user-friendly cost-effective fluorescence assay and by the ultra-sensitive SERS-based techniques. SERS microfluidic sensing platforms integrating plasmonic nanostructures will be developed, with a customization devoted to multiplexed analysis. The detection strategy will be also implemented with opticaltweezing, enabling the chemical-free creation and control of SERS-active sites in-liquid. Biochemical functionalization protocols for SERS substrates will be adapted to our PNA probes suitably designed to bound specific miR sequences. SERS-based platforms will provide high sensitivity while avoiding time-consuming chemical amplification steps, typical of polymerase chain reaction (PCR) assays. Large dynamic range (up to 4 decades) can be achieved, overcoming the performances of standard colorimetric analysis such ELISA. Finally, portability and moderate cost of the managed equipment will make these amplified spectroscopic analyses competitive with more complex tools such as real-time PCR and microarray technology.