Delayed Laboratory Response to COVID-19 Caused by Molecular Diagnostic Contamination.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.

Catalytic hairpin assembly induced dual signal enhancement for rapid detection of miRNA using fluorescence light-up silver nanocluster.

A novel method of catalytic hairpin assembly (CHA) induced dual signal enhancement is developed for rapid detection of miRNA based on fluorescence light-up silver nanocluster (Ag NC). By the hybridization of a hairpin DNA and a single-stranded DNA, a unique probe is firstly designed. In the terminals of this probe, DNA-Ag NCs can be formed and display very weak fluorescence. In the presence of the target miRNA, the reaction of CHA can be triggered, forming two kinds of double-stranded complexes, in which the terminal DNA-Ag NCs are in close proximity to G-rich overhangs and the fluorescent signal can be dramatically enhanced. Compared with many other enzyme-based amplification strategies, this one exhibits distinct advantages of simplicity in experimental operation and a rapid detection process (within 1 h). Moreover, this assay exhibits an excellent selectivity and is successfully applied in the detection of miRNAs in complex biological media, which confirms the reliability and practicality of this protocol.

Target-triggered dynamic hairpin assembly for signal amplification of microRNA and oncogenes and its application in live-cell imaging.

Based on target-triggered dynamic hairpin assembly (DHA) in both unidirectional and bilateral growth manners DNA nanobrushes are constructed, which realize sensitive and selective detection of short miRNA (miR-21) and long DNA (BRCA1), respectively. Moreover, the unidirectional DHA strategy is readily applied to in situ imaging of miR-21 in different live cells.

Bio-barcode technology for detection of Staphylococcus aureus protein A based on gold and iron nanoparticles.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL-1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL-1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL-1.

In situ fluorescence monitoring of diagnosis and treatment: a versatile nanoprobe combining tumor targeting based on MUC1 and controllable DOX release by telomerase.

We have constructed versatile drug-loaded nanoprobes capable of responding to both MUC1 and telomerase and achieving intracellular drug release. Besides, the synthesized drug-loaded nanoprobes can realize the in situ imaging observation of the whole process of nanoprobes targeting the tumor cell membrane, the transmembrane entering the cytoplasm and the release of DOX into the cell nucleus.

A novel method to detect the Mexican founder mutation BRCA1 ex9­12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes.

In 2015, according to the National Institute of Statistics and Geography (INEGI), malignant breast tumors were the first cause of cancer fatality in women (6,273 fatalities) in Mexico, whereas 2,793 fatalities in women were due to ovarian cancer. A total of 5­10% of breast cancer and 10­15% of ovarian cancer cases are caused by a hereditary breast­ovarian cancer syndrome, with mutations predominantly identified in the BRCA1 and BRCA2 genes. Recently, the Mexican founder mutation BRCA1 ex9­12del was identified (deletion of exons 9­12 with recombination between introns 8­12). This is the most frequently reported mutation in hereditary breast/ovarian cancer in Mexico. Current detection methods include end­point polymerase chain reaction (PCR) and Multiplex Ligation­dependent Probe Amplification (MLPA). In the present study a cheap, sensitive and fast detection method was developed based on quantitative PCR and two TaqMan® probes, one to detect the deletion (recombination region between introns 8 and 12), and the other one a region from exon 11. With this assay, 90 samples were able to be analyzed in 2 h using 2.5 ng of DNA/reaction at a cost of ~2­3 USD. This method is capable of detecting positive samples for DNA deletion and excluding negative ones. Therefore, the method proposed may be a useful high­throughput diagnostic option that could be useful in future association or prevalence studies that use large populations.

Fluorescence method for quickly detecting ochratoxin A in flour and beer using nitrogen doped carbon dots and silver nanoparticles.

In this paper, a FRET (Forster resonance energy transfer) based fluorescence method was developed for the quickly detection of ochratoxin A (OTA) in agricultural products (e.g., flour and beer). A highly fluorescent nitrogen doped carbon dots (CD) were served as energy donor, the DNA and MCH (6-mercapto-1-hexanol) modified Ag nanoparticles were served as energy acceptor in the FRET system. OTA can be detected in a concentration range between 10 and 5000 nM, the limit of detection is 8.7 nM. This method has three advantages: (1) an enhanced fluorescent intensity can be acquired by utilizing the nitrogen doped CD synthesized by one-step approach without sophisticated modification of nanoparticles; (2) OTA detection was accomplished quickly (less than 30 min) by using MCH as assistant molecule; (3) an extended OTA detection linear range was acquired, which may facilitate the OTA detection in real agricultural samples, and is helpful for solving food safety problems.

Luminescent quantum dots for miRNA detection.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are involved in nearly all developmental processes and human pathologies. MiRNAs are considered to be promising biomarkers, since their dysregulation correlates with the development and progress of many diseases. Short length, sequence homology among family members, susceptibility to degradation, and low abundance in total RNA samples make miRNA analysis a challenging task. Photoluminescent semiconductor nanoparticles (quantum dots, QDs) possess unique properties such as bright photoluminescence, photostability and narrow emission peaks, wide possibilities for surface modification and bioconjugation, which serve as the basis for the development of different analytical methods for variety of analytes. Relatively small size of QDs' and their narrow distribution are especially important for miRNA assay. The combination of QD-based biosensors with amplification techniques makes it possible to identify the target miRNA at a single-particle level with the detection limit at the attomolar scale. This review describes the principles of signal generation: direct intensity measurements, different "signal on" and "signal off" mechanisms as well as electro-chemiluminescence. Special attention is paid to the FRET-based techniques. According to our knowledge this is the first review related to QDs application for miRNA detection.

Sensitive and specific detection of microRNAs based on two-stage amplification reaction using molecular beacons as turn-on probes.

In this study, a rapid, sensitive, and specific assay for detecting miRNAs was developed based on a two-stage amplification reaction (TSAR) using molecular beacons (MBs) as turn-on probes. In the TSAR, different miRNAs can be converted to the same reporter oligonucleotides (Y), which can hybridize with the same MB. Therefore, in combination with specific templates, this method can be applied to multiplex miRNA detection by simply using the same MB. The loop region of the MB was screened by computer simulation methods. In particular, to improve the specificity of the MB in real sample analysis, the maximum similarity of the MB loop region to the human genome and human transcriptome is less than 70%. Two MBs were designed in this study. MB I, with nine flanking base pairs in its stem region, was used for real-time monitoring of the production of Y during the TSAR. MB II, with five flanking base pairs in its stem region, was used to detect the production of Y at the end of the TSAR. This assay exhibited high sensitivity with a limit of detection of 2.0 × 10-16M and 6.7 × 10-16M using MB I and MB II as turn-on probes, respectively. In addition, this assay can clearly discriminate single base differences in miRNA sequences, and the TSAR can be completed under isothermal conditions. Accordingly, the isothermal reaction conditions and simple fluorescence measurement can greatly contribute to the development of a fast point-of-care detection system.

Nucleic Acid Self-Assembly Circuitry Aided by Exonuclease III for Discrimination of Single Nucleotide Variants.

Robust and rapid discrimination of one base mutations in nucleic acid sequences is important in clinical applications. Here, we report a hybridization-based assay exploiting nucleic acid self-assembly circuitry and enzyme exonuclease III (Exo III) for the differentiation of single nucleotide variants (SNVs). This one-step approach combines the merits of discrimination power of competitive DNA hybridization probes (probe + sink) with catalytic amplification assisted by Exo III. The phosphorothioate bonds modified on a wild-type (WT) specific sink inhibit the Exo III digestion; thus, subsequent catalytic amplification magnifies only the intended SNV targets. The integrated assay exhibits improved SNV discrimination rather than hybridization probes relying solely on competition or amplification and enables SNV detection at 1% abundance. Two frequent cancer-driver mutation sequences (EGFR-L861Q, NRAS-Q61K) were tested. Our strategy allows simple sequence design and can easily adapt to multianalyte SNV detections.

Site-specific covalent capture of human O6-alkylguanine-DNA-alkyltransferase using single-stranded intrastrand cross-linked DNA.

A methodology is reported to conjugate human O6-alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O4-thymidine-alkylene-O4-thymidine and the (5'→3') O6-2'-deoxyguanosine-alkylene-O4-thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5'→3') O4-thymidine-alkylene-O6-2'-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

Quantification of microRNA by DNA-Peptide Probe and Liquid Chromatography-Tandem Mass Spectrometry-Based Quasi-Targeted Proteomics.

The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarkers. However, one of prerequisites for the application of miRNAs in clinical practice is to accurately profile their expression. Currently available assays normally require pre-enrichment, amplification, and labeling steps, and most of them are semiquantitative. In this study, we converted the signal of target miR-21 into reporter peptide by a DNA-peptide probe and the reporter peptide was ultimately quantified using LC-MS/MS-based targeted proteomics. Specifically, substrate peptide GDKAVLGVDPFR containing reporter peptide AVLGVDPFR and tryptic cleavage site (lysine at position 3) was first designed, followed by the conjugation with DNA sequence that was complementary to miR-21. The newly formed DNA-peptide probe was then hybridized with miR-21, which was biotinylated and attached to streptavidin agarose in advance. After trypsin digestion, the reporter peptide was released and monitored by a targeted proteomics assay. The obtained limit of quantification (LOQ) was 1 pM, and the detection dynamic range spanned ∼5 orders of magnitude. Using this assay, the developed quasi-targeted proteomics approach was applied to determine miR-21 level in breast cells and tissue samples. Finally, qRT-PCR was also performed for a comparison. This report grafted the strategy of targeted proteomics into miRNA quantification.

Sensitive Detection of Single-Nucleotide Mutation in the BRAF Mutation Site (V600E) of Human Melanoma Using Phosphate-Pyrene-Labeled DNA Probes.

A series of novel nucleotide phosphoramidites were rationally designed and synthesized and were then site-specifically incorporated in DNA oligonucleotide probes with pyrene-modified phosphate. These oligodeoxynucleotide (ODN) probes almost have no inherent fluorescence emission with pyrene modification at 3' phosphate of corresponding nucleotides as a result of the photoinduced electron-transfer quenching effect of nucleobases (thymidine ∼ cytidine > guanosine ≫ adenosine). However, strong fluorescence emission was observed only with the perfectly matched duplex for the probes with pyrene modified at 3' phosphate of thymidine and cytidine. These rationally designed ODN probes successfully worked as "turn on" fluorescence oligonucleotide sensors for single-nucleotide polymorphism (SNP) and were used for detecting a single BRAF mutation site (V600E) of human melanoma.

Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes.

The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH.

Identification of structure-activity relationships from screening a structurally compact DNA-encoded chemical library.

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.

Molecular design of an environmentally sensitive fluorescent nucleoside, 3-deaza-2'-deoxyadenosine derivative: distinguishing thymine by probing the DNA minor groove.

An environmentally sensitive fluorescent nucleoside containing a 3-deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3-naphthylethynylated 3-deaza-2'-deoxyadenosine ((3nz) A, 1) exhibited dual fluorescence emission from an intramolecular charge-transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength.

Highly sensitive and specific multiplexed microRNA quantification using size-coded ligation chain reaction.

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample.

New synthetic substrates of mammalian nucleotide excision repair system.

DNA probes for the studies of damaged strand excision during the nucleotide excision repair (NER) have been designed using the novel non-nucleosidic phosphoramidite reagents that contain N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nFlu) moieties. New lesion-imitating adducts being inserted into DNA show good substrate properties in NER process. Modified extended linear nFlu- and nAntr-DNA are suitable for estimation of specific excision activity catalysed with mammalian whole-cell extracts. The following substrate activity range was revealed for the model 137-bp linear double-stranded DNA: nAnt-DNA ≈ nFlu-DNA > Chol-DNA (Chol-DNA--legitimate NER substrate that contains non-nucleoside fragment bearing cholesterol residue). In vitro assay shows that modified DNA can be a useful tool to study NER activity in whole-cell extracts. The developed approach should be of general use for the incorporation of NER-sensitive distortions into model DNAs. The new synthetic extended linear DNA containing bulky non-nucleoside modifications will be useful for NER mechanism study and for applications.

ICON probes: synthesis and DNA methylation typing.

DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression, respectively. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis and may be a useful and effective index for cancer diagnosis. We have developed an artificial DNA probe that induces a methylation detection reaction of a target cytosine in a long DNA sequence (ICON probe). This artificial DNA allows the rapid detection of a methyl group attached at the C5 position of the target cytosine. In addition, there is no nonspecific cleavage of genomic DNA in this reaction. The ICON probe also facilitates the quantification of methylation at the target cytosine using a small amount of genomic DNA sample. This unit provides a procedure for synthesizing bipyridine-modified adenosine phosphoramidite and preparation of ICON probes. Additionally, the protocol for the methylation quantification experiments by quantitative PCR utilizing ICON probes is also presented.