Nucleic Acid Probes Research Articles

Detection of the level of DNMT1 based on self-assembled probe signal amplification technique in plasma

DNA (cytosine-5)-methyltransferase1 (DNMT1) is the most abundant DNA methyltransferase in somatic cells, and it plays an important role in the initiation, occurrence, and rehabilitation of tumors. Herein, we developed a novel strategy for the detection of the level of DNMT1 in human plasma using the self-assembled nucleic acid probe signal amplification technology. In this method, the DNMT1 monoclonal antibody (McAbDNMT1) was immobilized on carboxyl magnetic beads to form immunomagnetic beads and then captured DNMT1 specifically. After that, DNMT1 polyclonal antibody (PcAbDNMT1) and biotinylated sheep anti-rabbit IgG (sheep anti rabbit IgG-Biotin) were sequentially added into the system to react with DNMT1 and form biotinylated double antibody sandwich immunomagnetic beads. In the presence of the bridging medium streptavidin, the biotinylated double antibody sandwich immunomagnetic beads would form a complex with biotinylated poly-fluorescein (Biotin-poly FAM), and the fluorescence intensity of the complex was proportional to the concentration of DNMT1. Immunomagnetic beads can capture the target DNMT1 in the sample, and Biotin-poly FAM can realize signal amplification. Using these strategies, we got a linear range of the system for DNMT1 level detection was from 2 nmol/L to 200 nmol/L, and the limit of detection (LOD) was 0.05 nmol/L. The method was successfully applied for the determination of DNMT1 in human plasma with the recovery of 101.3–106.0%. Therefore, this method has the potential for the detection of DNMT1 level in clinical diagnosis.

Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip “fluorescent nucleic acid probe in droplets for bacterial sorting” (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.

Avoiding False Positive Signals: A Powerful and Reliable Au-Se Dual-Color Probe.

Nucleic acids as the important tumor markers play a crucial role in the identification of cancer. Various kinds of probes such as gold nanoparticles and graphene oxide have been explored to detect different nucleic acid markers. However, the existing probes are mostly used to detect a single tumor marker and susceptible to harsh conditions in the complex and dynamic physiological environment, which may lead to false positive results and greatly limit the sensing performance of the probe. Herein, a powerful and reliable Au-Se probe was developed for high-fidelity imaging of two cancer markers simultaneously in living cells. Compared with the traditional nucleic acid probe based on the Au-S bond, this probe was more stable against biological thiols and could effectively distinguish normal cells and cancer cells to avoid false positive results, which is more suitable for imaging in a complex physiological environment. This strategy will provide more valuable insights into designing and exploring novel biosensors in the future.

High Expression of microRNA-223 Indicates a Good Prognosis in Triple-Negative Breast Cancer.

PurposeMicroRNAs can influence many biological processes and have shown promise as cancer biomarkers. Few studies have focused on the expression of microRNA-223 (miR-223) and its precise role in breast cancer (BC). We aimed to examine the expression level of miR-223 and its prognostic value in BC.MethodsTissue microarray (TMA)-based miRNA detection in situ hybridization (ISH) with a locked nucleic acid (LNA) probe was used to detect miR-223 expression in 450 BC tissue samples. Overall survival (OS) and disease-free survival (DFS) were compared between two groups using the Kaplan-Meier method and Cox regression model.ResultsOS and DFS were prolonged in the high miR-223 expression group compared to the low miR-223 expression group (p < 0.0001 and p = 0.017, respectively), especially in patients with the triple-negative breast cancer (TNBC) subtype (p = 0.046 and p < 0.001, respectively). Univariate and multivariate Cox regression analyses revealed that TNM stage (p = 0.008), the molecular subtype (p = 0.049), and miR-223 (p < 0.001) were independently associated with OS and DFS. External validation was performed with the METABRIC and The Cancer Genome Atlas (TCGA) databases via online webtools and was consistent with the data described above.ConclusionsThis study provides evidence that high miR-223 expression at diagnosis is associated with improved DFS and OS for BC patients, especially those with the TNBC subtype. miR-223 is a valid and independent prognostic biomarker in BC.

Chemical binding of pyrrolidinyl peptide nucleic acid (acpcPNA-T9) probe with AuNPs toward label-free monitoring of miRNA-21: A novel biosensing platform for biomedical analysis and POC diagnostics.

miRNAs are attractive factors in cancer research studies due to their important roles for regulating of gene expression. Because of miRNA-21 expression surplus in many types of cancers, so accurate identification is important. Increasing efforts have caused different methods to improve the sensitivity and specificity of detection. Present study is an attempt to report a new electrochemical label-free PNA-based bioassay for detection of miRNA-21. In this study, gold electrode was modified by gold nanoparticles to improve a functional PNA-based biosensor. The EDS and field emission scanning electron microscope (FE-SEM) were used to detect fabrication of biosensor. The electrochemical behavior of sensor was evaluated after inserting of acpcPNA probes and miRNA-21 on the stucture of electrode and analyzed essential parameters such as various concentration of target miRNA, hybridization time, reproducibility, stability, and applicability. The results of study demonstrated that engineered biosensor was successfully fabricated. The findings showed the highest amount of current in 5 minutes hybridization time, with suitable reproducibility and stability. This innovative miRNA-based biosensor presents a sensitive and specific method in fast and may be lab-on chip assay in future.

Temperature-Enhanced mcr-1 Colistin Resistance Gene Detection with Electrochemical Impedance Spectroscopy Biosensors.

Antibiotic resistance is now one of the biggest threats humankind is facing, as highlighted in a declaration by the General Assembly of the United Nations in 2016. In particular, the growing resistance rates of Gram-negative bacteria cause increasing concerns. The occurrence of the easily transferable, plasmid-encoded mcr-1 colistin resistance gene further worsened the situation, significantly enhancing the risk of the occurrence of pan-resistant bacteria. There is therefore a strong demand for new rapid molecular diagnostic tests for the detection of mcr-1 gene-associated colistin resistance. Electrochemical impedance spectroscopy (EIS) is a well-suited method for rapid antimicrobial resistance detection as it enables rapid, label-free target detection in a cost-efficient manner. Here, we describe the development of an EIS-based mcr-1 gene detection test, including the design of mcr-1-specific peptide nucleic acid probes and assay specificity optimization through temperature-controlled real-time kinetic EIS measurements. A new flow cell measurement setup enabled for the first time detailed real-time, kinetic temperature-controlled hybridization and dehybridization studies of EIS-based nucleic acid biosensors. The temperature-controlled EIS setup allowed single-nucleotide polymorphism discrimination. Target hybridization at 60 °C enhanced the perfect match/mismatch (PM/MM) discrimination ratio from 2.1 at room temperature to 3.4. A hybridization and washing temperature of 55 °C further increased the PM/MM discrimination ratio to 5.7 by diminishing the mismatch signal during the washing step while keeping the perfect match signal. This newly developed mcr-1 gene detection test enabled the direct, specific label, and amplification-free detection of mcr-1 gene harboring plasmids from Escherichia coli.

Highly Sensitive Detection of Low-Abundance BRAF V600E Mutation in Fine-Needle Aspiration Samples by Zip Recombinase Polymerase Amplification.

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.

Rational Design of a Dual-Layered Metal-Organic Framework Nanostructure for Enhancing the Cell Imaging of Molecular Beacons.

As the most popular nucleic acid probe, molecular beacons (MBs) can selectively light up endogenous RNA targets without specific treatment. However, the poor cell permeability and unsatisfied intracellular stability of MBs significantly restricted their detection performance. Herein, we report the encapsulation of MB within a dual-layered metal-organic framework nanostructure UiO66-ZIF8 for enhanced cell imaging. UiO66-NH2 nanoparticles were synthesized as the template for MB loading, and the ZIF-8 shell was further coated on the surface of UiO66-MB to ensure its stability and lysosomal escape effect. Taking multidrug-resistant (MDR1) mRNA as a model target, MBs loaded within UiO66-ZIF8 showed an improved lysosomal escape effect compared with MB absorbed on UiO66-NH2. Therefore, efficient and accurate intracellular MDR1 mRNA imaging was realized with UiO66-MB-ZIF8. This work presented a new method for the rational regulation of the intracellular fate of MOF-based nanoprobes and will facilitate the further development of hierarchical MOF nanoprobes for analytical applications.

Development of a real-time PCR assay using locked nucleic acid probe for the detection of octopine, nopaline, and vitopine strains of tumorigenic Allorhizobium vitis in grapevines

Crown gall disease caused by Allorhizobium vitis is one of the most destructive diseases in grapevine cultivation from an economical perspective. The bacterial pathogen exists systemically in grapevines and spreads through cuttings used for propagation material obtained from grapevines, living vines, and mother blocks, which poses a high risk for certified grapevine nursery material cultivation, nursery industry, and growers. Therefore, in this study, real-time PCR-based methods were developed for effectively and accurately determining the presence and the density of the bacterial pathogen in grapevine nursery material and the data used for indexing programs. Octopine-, nopaline-, and vitopine-catabolizing A. vitis strain-specific primer and locked nucleic acid (LNA) probe sets were determined depending on the ocs, nos, vis, vitopine iaaM, and vitopine virD2 genes. Primer-probe sets were then used to amplify the octopine synthase gene (62 bp) from octopine strains, the nopaline synthase gene (78 bp) from nopaline strains, and the vitopine synthase gene (60 bp) from vitopine strains, as well as the vitopine iaaM gene (60 bp) and the vitopine virD2 gene (66 bp) of A. vitis. The detection of A. vitis strains from bacterial suspension or extracted plant sap could be achieved within a short time (i.e., 20–25 min) with real-time PCR-based assays using the designed primer and probe sets. For the real-time PCR-based methods, the bacterial cell sensitivity threshold was defined as sensitivity threshold for one bacterial cell and at the DNA level, which was 1.4–1.9 fg. In real-time PCR-based methods, the corresponding PCR efficiencies for octopine and nopaline strains were 97.12% and 97.87%, respectively and for vitopine strains were 94.39%, 96.76%, and 99.01%. In this study, a new real-time PCR-based method was developed that uses the LNA probe, and different opine strains of A. vitis were detected and quantified from vegetative materials of infected grapevine.

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci.

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normalcells and in disease progression such as with cancer.

A novel approach for microRNA in situ hybridization using locked nucleic acid probes

Identification of target tissue microRNAs (miR) using in situ hybridization (ISH), with digoxigenin-labeled locked nucleic acid (LNA) probes, is influenced by preanalytic parameters. To determine the best retrieval method for common microRNAs, a multiblock composed of paraffin-embedded tonsil, cervix, placenta, and hyperplastic prostate tissue were included. Tissue were fixed in 10% formalin in a range of 5–144 hours (h). Cut sections (5 μm) from the multiblock were subjected to combinations of pretreatment procedures: variable periods of proteinase K (PK) digestion or Heat-induced microRNA Retrieval (HmiRR) using target retrieval solution (TRS) pH 6.1 or 9, with or without enzymatic treatment (pepsin). Results for the overall categories: TRS pH 9 versus PK; p = 2.9e−23, TRS pH 9 versus TRS pH 6.1; p = 1.1e−14, TRS pH 6.1 versus PK; p = 2.9e−03. A long fixation time, resulted in the best microRNA preservation and staining intensity (long vs. short: p = 3.5e−47, long vs. moderate: p = 1.6e−44, moderate vs. short: p = 4.3e−16), was enhanced using HmiRR TRS pH 9 with or without pepsin providing high sensitivity and specificity. These observations conflict with other ISH techniques (e.g., messenger ribonucleic acid), which typically require shorter fixation periods, and therefore, further studies are warranted.

Bioconjugation of a PNA Probe to Zinc Oxide Nanowires for Label-Free Sensing.

Zinc oxide nanowires (ZnONWs) are largely used in biosensing applications due to their large specific surface area, photoluminescence emission and electron mobility. In this work, the surfaces of ZnONWs are modified by covalent bioconjugation of a peptidic nucleic acid (PNA) probe whose sequence is properly chosen to recognize a complementary DNA (cDNA) strand corresponding to a tract of the CD5 mRNA, the main prognostic marker of chronic lymphatic leukemia. The interaction between PNA and cDNA is preliminarily investigated in solution by circular dichroism, CD melting, and polyacrylamide gel electrophoresis. After the immobilization of the PNA probe on the ZnONW surface, we demonstrate the ability of the PNA-functionalized ZnONW platform to detect cDNA in the μM range of concentration by electrical, label-free measurements. The specificity of the sensor is also verified against a non-complementary DNA sequence. These preliminary results highlight the potential application of PNA-bioconjugated ZnONWs to label-free biosensing of tumor markers.

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

Temperature-robust and ratiometric G-quadruplex proximate DNAzyme assay for robustly monitoring of uranium pollution and its microbial biosorbents screening

Uranium pollution in environment and food chain is a serious threat to public security and human health. Herein, we proposed a temperature-robust, ratiometric, and label-free bioassay based on G-quadruplex proximate DNAzyme (G4DNAzyme), accommodating us to precisely monitor uranium pollution and biosorption. The proximity of split G-quadruplex probes was proposed to sense UO22+-activated DNAzyme activity, thus eliminating the use of chemically labeled nucleic acid probes. And the simultaneous monitoring of G-quadruplex and double-stranded structures of DNAzyme probes contributed to a ratiometric and robust detection of UO22+. Particularly, the separation of enzymatic digestion and fluorescence monitoring endued a robust and highly responsive detection of UO22+ upon the temperature of enzymatic digestion process ranged from 18° to 41 °C. Consequently, G4DNAzyme assay allowed a robust, label-free and ratiometric quantification of uranium. We demonstrated the feasibility of G4DNAzyme assay for estimating uranium pollution in water and aquatic product samples. Ultimately, G4DNAzyme assay was adopted to serve as the platform to screen bacterial species and conditions for uranium biosorption, promising its roles in uranium associated biosafety control.

Early detection of T-cell lymphoma with T follicular helper phenotype by RHOA mutation analysis.

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma with T follicular helper phenotype (PTCL-TFH) are a group of complex clinicopathological entities that originate from T follicular helper cells and share a similar mutation profile. Their diagnosis is often a challenge, particularly at an early stage, because of a lack of specific histological and immunophenotypic features, paucity of neoplastic T cells and prominent polymorphous infiltrate. We investigated whether the lymphoma-associated RHOA Gly17Val (c.50G>T) mutation, occurring in 60% of cases, is present in the early “reactive” lesions, and whether mutation analysis could help to advance the early diagnosis of lymphoma. The RHOA mutation was detected by quantitative polymerase chain reaction with a locked nucleic acid probe specific to the mutation, and a further peptide nucleic acid clamp oligonucleotide to suppress the amplification of the wild-type allele. The quantitative polymerase chain reaction assay was highly sensitive and specific, detecting RHOA Gly17Val at an allele frequency of 0.03%, but not other changes in Gly17, nor in 61 controls. Among the 37 cases of AITL and PTCL-TFH investigated, RHOA Gly17Val was detected in 62.2% (23/37) of which 19 had multiple biopsies including preceding biopsies in ten and follow-up biopsies in 11 cases. RHOA Gly17Val was present in each of these preceding or follow-up biopsies including 18 specimens that showed no evidence of lymphoma by combined histological, immunophenotypic and clonality analyses. The mutation was seen in biopsies 0-26.5 months (mean 7.87 months) prior to the lymphoma diagnosis. Our results show that RHOA Gly17Val mutation analysis is valuable in the early detection of AITL and PTCL-TFH.

Fluorometric Viral miRNA Nanosensor for Diagnosis of Productive (Lytic) Human Cytomegalovirus Infection in Living Cells.

A human cytomegalovirus (HCMV) causes a persistent asymptomatic infection in healthy individuals and possesses unexpected dangers to newborn babies, immunocompromised people, and organ transplant recipients because of stealth transmission. Thus, an early and accurate diagnosis of HCMV infection is crucial for prevention of unexpected transmission and progression of the severe diseases. The standard method of HCMV diagnosis depends on serology, antigen test, and polymerase chain reaction-based nucleic acid detection, which have advantages for each target molecule. However, the serological test for an antibody is an indirect method assuming the past virus infection, and antigen and viral nucleic acid testing demand laborious, complex multistep procedures for direct virus detection. Herein, we present an alternative simple and facile fluorometric biosensor composed of a graphene oxide nanocolloid and fluorescent peptide nucleic acid (PNA) probe to detect the HCMV infection by simply monitoring the virally encoded microRNA as a new biomarker of lytic virus infection. We verify the sensing of HCMV-derived microRNA accumulated within 72 h after HCMV infection and examine the diagnosis of HCMV in living cells. We proceed with the time course and concentration-dependent investigation of hcmv-miRNA sensing in living cells as a direct method of HCMV detection at the molecular level on the basis of an intracellular hcmv-miRNA expression profile and graphene oxide nanocolloid-based simple diagnostic platform. The fluorometric biosensor enables the sequence-specific binding to the target HCMV miRNAs in HCMV-infected fibroblasts and shows the quantitative detection capability of HCMV infection to be as low as 4.15 × 105 immunofluorescence focus unit (IFU)/mL of the virus titer at 48 h post-infection with picomolar sensitivity for HCMV miRNA.

Scalable Logic Circuits with Multiple Outputs and an Automatic Reset Function Based on DNAzyme-Mediated Branch Migration.

A scalable logic platform made up of multilayer DNA circuits was constructed using Pb2+, Cu2+, and Zn2+ as the three inputs and three different fluorescent signals as the outputs. DNAzyme-guided cyclic cleavage reactions and DNA toehold-mediated strand branch migration were utilized to organize and connect nucleic acid probes for building the high-level logic architecture. The sequence communications between each circuit enable the logic network to work as a keypad lock, which is an information protection model at the molecular level. The multi-output mode was used to monitor the gradual unlocking process of the security system, from which one can determine which password is correct or not immediately. The autocatalytic cleavage of DNAzyme makes the biocomputing circuit feasible to realize the reset function automatically without external stimuli. Importantly, the logic platform is robust and can work effectively even in complex environmental samples.

High-Coverage Nucleic Acid Probes for Distinguishing SARS-COV-2 from Influenza

There is an urgent need for rapid, accurate, and ultrasensitive diagnostics to identify respiratory viruses. Amplification-free detection of nucleic acids provides a promising technique for rapid diagnosis of viral infections. The objective of this study was to design, characterize, and optimize mutation-resistant molecular beacon probes for the detection of influenza versus SARS-CoV-2 viruses. High-coverage probe cocktails were computationally designed using our “fast evaluation of viral emerging risks” (FEVER) pipeline.

Pyrene-Modified Guanine Cluster Probes Forming DNA/RNA Hybrid Three-Way Junctions for Imaging of Intracellular MicroRNAs.

MicroRNAs (miRNAs) regulate gene expression in cells; high levels of expression are associated with various cancers. In this paper, we describe PyA-modified nucleic acid probes that can detect intracellular miRNAs by forming DNA/RNA hybrid three-way junction structures containing a fluorescent scaffold-a so-called G-cluster. This G-cluster featured two mismatched strands, four guanine residues, and one fluorescent adenine residue having a pyrene moiety covalently connected at the 8-position through an acetylene linker. The scaffold underwent a dramatic shift in its emission wavelength when two mismatched strands formed a duplex, similar to the behavior of an adenine pentad system (A-cluster). We applied the G-cluster scaffold in a three-way junction system to probe for miRNAs; its red-shifted fluorescence intensity and stability were greater than those reported previously for A-cluster three-way junction probes. Furthermore, confocal microscopy of cancer cell lines revealed bright fluorescence emissions in response to the miRNAs in the cells. Thus, this system can be applied intracellularly as a potential fluorescent probe for the detection of various biologically important nucleic acids.

Discovery and Proof-of-Concept Study of Nuclease Activity as a Novel Biomarker for Breast Cancer Tumors

Simple SummaryA diagnostic biomarker for the detection of breast cancer remains an unmet clinical need despite decades of intensive research efforts. Herein, we describe, for the first time, the use of nuclease activity as a biomarker to discriminate between healthy and cancer biopsy samples. We have identified a panel of three nucleic acid probes able to target nucleases derived from breast cancer tumors with high sensitivity and specificity. These results are in good agreement with histopathological analysis as the diagnostic gold standard. Moreover, these findings support nuclease activity as a potential adjacent diagnostic tool and shed light on the use of nuclease activity as a detection biomarker in breast cancer.Breast cancer is one of the most common pathologies diagnosed in the clinical practice. Despite major advancements in diagnostic approaches, there is no widely accepted biomarker in the clinical practice that can diagnose breast malignancy. Confirmatory diagnosis still relies on the pathological assessment of tissue biopsies by expert pathologists. Thus, there is an unmet need for new types of biomarkers and novel platform technologies that can be easily and robustly integrated into the clinic and that can assist pathologists. Herein, we show that nuclease activity associated to malignant tumors can be used as a novel biomarker in breast cancer, which can be detected via specific degradation of nucleic acid probes. In this study we have identified a set of three chemically modified nucleic acid probes that can diagnose malignancy in biopsy samples with high accuracy (89%), sensitivity (82%) and specificity (94%). This work represents a breakthrough for the potential clinical use of nuclease activity as biomarker, which can be detected via nucleic acids probes, for the clinical diagnosis of malignancy in breast tissue biopsies. This platform technology could be readily implemented into the clinic as adjunct to histopathological diagnostic.