Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum

MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 μl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.

Application of Nanotechnology for Sensitive Detection of Low-Abundance Single-Nucleotide Variations in Genomic DNA: A Review

Single-nucleotide polymorphisms (SNPs) are the simplest and most common type of DNA variations in the human genome. This class of attractive genetic markers, along with point mutations, have been associated with the risk of developing a wide range of diseases, including cancer, cardiovascular diseases, autoimmune diseases, and neurodegenerative diseases. Several existing methods to detect SNPs and mutations in body fluids have faced limitations. Therefore, there is a need to focus on developing noninvasive future polymerase chain reaction (PCR)–free tools to detect low-abundant SNPs in such specimens. The detection of small concentrations of SNPs in the presence of a large background of wild-type genes is the biggest hurdle. Hence, the screening and detection of SNPs need efficient and straightforward strategies. Suitable amplification methods are being explored to avoid high-throughput settings and laborious efforts. Therefore, currently, DNA sensing methods are being explored for the ultrasensitive detection of SNPs based on the concept of nanotechnology. Owing to their small size and improved surface area, nanomaterials hold the extensive capacity to be used as biosensors in the genotyping and highly sensitive recognition of single-base mismatch in the presence of incomparable wild-type DNA fragments. Different nanomaterials have been combined with imaging and sensing techniques and amplification methods to facilitate the less time-consuming and easy detection of SNPs in different diseases. This review aims to highlight some of the most recent findings on the aspects of nanotechnology-based SNP sensing methods used for the specific and ultrasensitive detection of low-concentration SNPs and rare mutations.

Detection of Single Nucleotide Polymorphisms by Fluorescence Embedded Dye SYBR Green I Based on Graphene Oxide

The detection of single nucleotide polymorphisms (SNPs) is of great significance in the early diagnosis of diseases and the rational use of drugs. Thus, a novel biosensor based on the quenching effect of fluorescence-embedded SYBR Green I (SG) dye and graphene oxide (GO) was introduced in this study. The probe DNA forms a double helix structure with perfectly complementary DNA (pcDNA) and 15 single-base mismatch DNA (smDNA) respectively. SG is highly intercalated with perfectly complementary dsDNA (pc-dsDNA) and exhibits strong fluorescence emission. Single-base mismatch dsDNA (SNPs) has a loose double-stranded structure and exhibits poor SG intercalation and low fluorescence sensing. At this time, the sensor still showed poor SNP discrimination. GO has a strong effect on single-stranded DNA (ssDNA), which can reduce the fluorescence response of probe DNA and eliminate background interference. And competitively combined with ssDNA in SNPs, quenching the fluorescence of SG/SNP, while the fluorescence value of pc-dsDNA was retained, increasing the signal-to-noise ratio. At this time, the sensor has obtained excellent SNP resolution. Different SNPs detect different intensities of fluorescence in the near-infrared region to evaluate the sensor's identification of SNPs. The experimental parameters such as incubation time, incubation temperature and salt concentration were optimized. Under optimal conditions, 1 nM DNA with 0–10 nM linear range and differentiate 5% SNP were achieved. The detection method does not require labeling, is low cost, simple in operation, exhibits high SNP discrimination and can be distinguished by SNP at room temperature.

Target-mediated base-mismatch initiation of a non-enzymatic signal amplification network for highly sensitive sensing of Hg2+

Target-triggered initiation of a non-enzymatic signal amplification network leads to highly sensitive fluorescence detection of Hg2+.

Understanding Rad51 function is a prerequisite for progress in cancer research

The human protein Rad51 is double-edged in cancer contexts: on one hand, preventing tumourigenesis by eliminating potentially carcinogenic DNA damage and, on the other, promoting tumours by introducing new mutations. Understanding mechanistic details of Rad51 in homologous recombination (HR) and repair could facilitate design of novel methods, including CRISPR, for Rad51-targeted cancer treatment. Despite extensive research, however, we do not yet understand the mechanism of HR in sufficient detail, partly due to complexity, a large number of Rad51 protein units being involved in the exchange of long DNA segments. Another reason for lack of understanding could be that current recognition models of DNA interactions focus only on hydrogen bond-directed base pair formation. A more complete model may need to include, for example, the kinetic effects of DNA base stacking and unstacking (‘longitudinal breathing’). These might explain how Rad51 can recognize sequence identity of DNA over several bases long stretches with high accuracy, despite the fact that a single base mismatch could be tolerated if we consider only the hydrogen bond energy. We here propose that certain specific hydrophobic effects, recently discovered destabilizing stacking of nucleobases, may play a central role in this context for the function of Rad51.

Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy

Circulating exosomal microRNA (miR) represents a new class of blood-based biomarkers for cancer liquid biopsy. The detection of miR at a very low concentration and with single-base discrimination without the need for sophisticated equipment, large volumes, or elaborate sample processing is a challenge. To address this, we present an approach that is highly specific for a target miR sequence and has the ability to provide “digital” resolution of individual target molecules with high signal-to-noise ratio. Gold nanoparticle tags are prepared with thermodynamically optimized nucleic acid toehold probes that, when binding to a target miR sequence, displace a probe-protecting oligonucleotide and reveal a capture sequence that is used to selectively pull down the target-probe–nanoparticle complex to a photonic crystal (PC) biosensor surface. By matching the surface plasmon-resonant wavelength of the nanoparticle tag to the resonant wavelength of the PC nanostructure, the reflected light intensity from the PC is dramatically and locally quenched by the presence of each individual nanoparticle, enabling a form of biosensor microscopy that we call Photonic Resonator Absorption Microscopy (PRAM). Dynamic PRAM imaging of nanoparticle tag capture enables direct 100-aM limit of detection and single-base mismatch selectivity in a 2-h kinetic discrimination assay. The PRAM assay demonstrates that ultrasensitivity (<1 pM) and high selectivity can be achieved on a direct readout diagnostic.

Single-Step FRET-Based Detection of Femtomoles DNA

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

Downregulation of TdT Expression through Splicing Modulation by Antisense Peptide Nucleic Acid (PNA)

Background and Objective: Antisense oligonucleotides are able to modulate splicing patterns and offer therapeutic intervention for cancer and other diseases. Considering TdT potential as a target in cancer therapy, the present study aimed to investigate splicing alteration of TdT pre-mRNA in Molt-4 cells using peptide nucleic acid (PNA) octaarginine and cholic acid conjugates. Method: We examined 16 mer PNAs targeting 5' and 3' junctions of intron 7 and addressed their mRNA splicing modulation effects using RT-PCR analysis. We also tested corresponding 2-base mismatch PNAs to confirm the sequence specificity. In addition, protien level of TdT, apoptosis induction and cell viability rate were analysed. Results: PCR analysis showed that full match PNAs could modulate the splicing process, thereby producing a longer mRNA still including intron 7. PCR results also implied exon 7 skipping. In addition, reduced level of TdT protein in Molt-4 cells was observed. Downregulation of TdT level in PNA treated cells was accompanied by an increased rate of apoptosis and decreased the level of cell survival. Conclusion: PNA-mediated splicing modulation can specifically downregulate TdT expression. TdT dowregulation results in apoptosis induction and reduced cell survival in Molt-4 cells. These observations could draw more attentions to develop PNA based strategies for TdT suppression and consequent apoptosis induction in acute lymphoblastic leukemia.