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Although circulating tumour DNA (ctDNA)-based next-generation sequencing (NGS) is a less invasive method for assessing ESR1 mutations that are essential mechanisms of endocrine therapy resistance in patients with oestrogen receptor-positive breast cancer, adequate amounts of DNA are required to assess polyclonal ESR1 mutations. By combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay, we have developed a novel detection system to screen for polyclonal ESR1 mutations in ctDNA. A validation assay was prospectively performed on clinical samples and compared with the NGS results. The PNA-LNA PCR clamp assay was validated using six and four blood samples in which ESR1 mutations were detected by NGS and no mutations were detected, respectively. The PNA-LNA assay results were comparable with those of NGS. We prospectively assessed the concordance between the PNA-LNA PCR clamp method and NGS. Using the PNA-LNA PCR clamp method, ESR1 mutations were detected in 5 out of 18 samples, including those in which mutations were not detected by NGS due to small amounts of ctDNA. The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for polyclonal ESR1 mutation detection in the ctDNA of patients with breast cancer.

EGFR mutation testing has become an essential determination to decide treatment options for NSCLC. The mutation analysis is often conducted in samples with low percentage of tumour cells from primary tumour biopsies. There is very little evidence that samples from metastatic tissues are suitable for EGFR testing. We had evaluated the frequency of EGFR mutations with three highly sensitive PCR techniques in formalin-fixed, paraffin-embedded samples of 143 NSCLC patients with central nervous system (CNS) metastases. 32 corresponding primary tumours were also examined. We used PCR followed by DNA fragments length analysis (FLA), ASP–PCR and PNA–LNA PCR clamp techniques. We found 9 (6.29 %) EGFR gene mutations in CNS samples: 3 (2.1 %) in exon 19 and 6 (4.2 %) in exon 21. The full concordance between CNS metastases and primary tumour samples was observed. PCR followed by DNA–FLA and PNA–LNA PCR clamp were sensitive enough to detect exon 19 deletions. Two mutations in exon 21 were detected by ASP–PCR only, one L858R substitution was detected only by PNA–LNA PCR clamp. With respect to sensitivity, PCR followed by DNA–FLA achieved a level of detection of at least 10 % of mutated DNA for exon 19 deletion, as for ASP–PCR it was at least 5 % of mutated DNA for L858R substitution. Higher sensitivity of 1 % of mutated DNA was achieved by PNA–LNA PCR clamp technique for both mutations. The use of different methodological techniques authenticates the negative result of molecular tests.

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.

MicroRNAs (miRNAs) have emerged as pivotal regulators in cancer biology, influencing tumorigenesis, progression, and resistance to therapy. Their ability to modulate multiple oncogenic and tumor-suppressive pathways positions them as promising therapeutic tools or targets. This review examines the dual role of miRNAs in solid and hematological malignancies, starting from their dysregulation in various cancer types. Therapeutic approaches, including miRNA replacement and inhibition strategies, are discussed alongside innovative delivery systems such as lipid nanoparticles and exosomes. Despite their transformative potential, challenges persist, including off-target effects, immune activation, and delivery inefficiencies. Recent clinical trials demonstrate both progress and hurdles, underscoring the need for advanced strategies to optimize specificity and minimize toxicity. This review provides an updated comprehensive overview of the current landscape of miRNA-based therapies under early clinical investigation and explores future directions for integrating these approaches into precision oncology.

G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that have gathered significant interest in medicinal chemistry over the past two decades due to their unique structural features and potential roles in a variety of biological processes and disorders. Traditionally, research efforts have focused on stabilizing G4s, while in recent years, the attention has progressively shifted to G4 destabilization, unveiling new therapeutic perspectives. This review provides an in-depth overview of recent advances in the development of small molecules, starting with the controversial role of TMPyP4. Moreover, we described effective metal complexes in addition to G4-disrupting small molecules as well as good G4 stabilizing ligands that can destabilize G4s in response to external stimuli. Finally, we presented antisense strategies as a promising approach for destabilizing G4s, with a particular focus on 2′-OMe antisense oligonucleotide, peptide nucleic acid, and locked nucleic acid. Overall, this review emphasizes the importance of understanding G4 dynamics as well as ongoing efforts to develop selective G4-unfolding strategies that can modulate their biological function and therapeutic potential.

Emerging evidence suggests that exosomal microRNAs are potential biomarkers for the early diagnosis and prognostic assessment of tumor. Here, we design a strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy for highly specific and sensitive electrochemical sensing of exosomal microRNAs. In the presence of exosomal miRNA-21, a locked nucleic acid (LNA)-labeled toehold mediated strand displacement reaction (TMSDR) is initiated, releasing output P2 to trigger the subsequent RCA reaction by hybridizing with the C-rich circular template. Then the obtained G-rich RCA products can bind to the probe anchored on the surface of gold electrode and generate G-quadruplex conformations. Based on the TMSDR-triggered G-quadruplex/RCA strategy, the detection limit of this electrochemical biosensor is down to 2.75 fM. Moreover, our biosensor exhibits excellent repeatability, stability, and high consistency compared to RT-PCR for clinical detection. In conclusion, this assay is expected to provide a hopeful strategy for the early non-invasive diagnosis and prognostic estimation of cancer. Graphical abstract Schematic illustration of electrochemical sensing of exosomal microRNAs based on strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy.

Locked nucleic acid (LNA™) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination ( Tm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA-DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.

DNA circuits based on toehold-mediated DNA strand displacement reaction are powerful tools owing to their programmability and predictability. However, performance and practical application of the circuits are greatly restricted by leakage, which refers to the fact that there is no input (invading strand) in the circuit, and the output signal is still generated. Herein, we constructed locked nucleic acids-based DNA circuits with ultra-low leakage. High binding affinity of LNA (locked nucleic acid)-DNA/LNA suppressed the leakage by inhibiting the breathing effect. Based on the strategy, we have built various low-leakage DNA circuits, including translator circuit, catalytic hairpin assembly (CHA) circuit, entropy-driven circuit (EDC), and seesaw circuit. More importantly, our strategy would not affect the desired main reactions: The output signal remained above 85% for all tested circuits, and the signal-to-noise ratios were elevated to 148.8-fold at the most. We believe our strategy will greatly promote the development and application of DNA circuits-based DNA nanotechnology.