Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18 for short DNA and 54 for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA fieldProject FUNMOL of the EECC (FP7-NMP-213382-2); the Spanish Ministry of Education (BFU2007-63287, CTQ2010-20541, TEC2009-08729 and AGL2010-17181); the project Microplex from CIBER-BBN (CIBER-BBN [The Biomedical Research Networking center in Bioengineering, Biomaterials and Nanomedicine] which is an initiative funded by the VI National R&D&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.); the Generalitat de Catalunya (2009/SGR/208) and M. Botin Foundation. Funding for open access publication charges: Spanish Ministry of Education: CTQ2010-20541, AGL2010-17181, TEC2009-0872