So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored.

Challenges in reliable nucleic acid detection are manifold. The major ones are related to false positive or negative signals due to a lack of target specificity in detection and to low sensitivity, especially when a plethora of background sequences are present that can mask the specific recognition signal. Utilizing designed synthetic nucleic acids that are commonly called xeno nucleic acids could offer potential routes to meeting such challenges. In this article, we present the general framework of nucleic acid detection, especially for nanoscale applications, and discuss how and why the xeno nucleic acids could be truly an alternative to the DNA probes. Two specific cases, locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are nuclease-resistant and can form thermally stable duplexes with DNA, are addressed. It is shown that the relative ease of the conformationally rigid LNA probe to be oriented upright on the substrate surface and of the nonionic PNA probe to result into high probe density assists in their use in nanoscale nucleic acid recognition. It is anticipated that success with these probes may lead to important developments such as PCR-independent approaches where the major aim is to detect a small number of target sequences present in the analyte medium.

A number of reports have been made in recent times on label-free detection of nucleic acid sequences. However, most of these studies deal with ensemble measurements, therefore lacking in molecular level resolution. These assays have usually employed ssDNA sensor probes, and often suffered from problems of irreproducibility and poor sequence-selectivity. Herein, the applicability of surface-anchored single stranded locked nucleic acid (ssLNA) probes has been assessed in the detection of target DNA sequences, as an alternative to the DNA-based assay. Importantly, the effectiveness of the LNA-based assay in identifying different types of single nucleobase mismatches has been tested. Since the duplex melting temperature is an indicator of duplex stability, the ensemble on-surface Tm values of the surface-confined LNA-DNA duplexes have been compared to the duplex unbinding force values obtained from atomic force spectroscopy (AFS) experiments. A common mismatch discrimination pattern elicited by both the ensemble and the molecular level AFS approach could be identified. Apart from quantitative delineation of the different types of mismatches, the label-free AFS analysis confirms different degrees of efficiency of the purine and pyrimidine bases, present on the LNA backbone, in discriminating different nucleobase mismatch types. Importantly, the LNA-based AFS analysis can distinguish between the disease-relevant gene fragments, e.g., multidrug-resistant Mycobacterium tuberculosis (MTB) mutation, and the wild type. Since LNA probes are nuclease-resistant, these findings could potentially pave way to diagnostic applications of the LNA-based AFS assay.

We have developed a label-free approach for direct detection of gene mutations using free-energy values that are derived from single-molecule force spectroscopy (SMFS)-based nucleic acid unbinding experiments. From the duplex unbinding force values acquired by SMFS, the force-loading-rate-independent Gibbs free-energy values were derived using Jarzinsky’s equality treatment. Because it provides molecule-by-molecule information, this approach is a major shift compared to the earlier reports on label-free detection of DNA sequences, which are mostly based on ensemble level data. We tested our approach in the disease model framework of multiple drug-resistant tuberculosis using the nuclease-resistant and conformationally rigid locked nucleic acid probes that are a robust and efficient alternative to the DNA probes. All of the major mutations in Mycobacterium tuberculosis (MTB), as relevant to MTB’s resistance to the first-line anti-TB drugs rifampicin and isoniazid, could be identified, and the wild type could be discriminated from the most prevalent mutation and the most prevalent mutation from the less occurring ones. Our approach could also identify DNA sequences (45 mer), having overhang stretches at different positions with respect to the complementary stretch. Probably for the first time, the findings show that free-energy-based detection of gene mutations is possible at molecular resolution.

Graphical abstract