Chimeric Probe Research Articles

Collagen Anchoring Protein-Nucleic Acid Chimeric Probe for In Situ In Vivo Mapping of a Tumor-Specific Protease.

In situ analysis of biomarkers in the tumor microenvironment (TME) is important to reveal their potential roles in tumor progression and early diagnosis of tumors but remains a challenge. In this work, a bottom-up modular assembly strategy was proposed for a multifunctional protein-nucleic chimeric probe (PNCP) for in situ mapping of cancer-specific proteases. PNCP, containing a collagen anchoring module and a target proteolysis-responsive isothermal amplification sensor module, can be anchored in the collagen-rich TME and respond to the target protease in situ and generate amplified signals through rolling cycle amplification of tandem fluorescent RNAs. Taking matrix metalloproteinase 2 (MMP-2), a tumor-associated protease, as the model, the feasibility of PNCP was demonstrated for the in situ detection of MMP-2 activity in 3D tumor spheroids. Moreover, in situ in vivo mapping of MMP-2 activity was also achieved in a metastatic solid tumor model with high sensitivity, providing a useful tool for evaluating tumor metastasis and distinguishing highly aggressive forms of tumors.

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells.

The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other off-target interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric d/l-DNA architecture that exploits the bioorthogonal properties of mirror-image l-DNA. We show that chimeric BER probes have excellent stability within living cells, where they were successfully employed to monitor relative BER activity, evaluate the efficiency of small molecule BER inhibitors, and study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.

A Single Multiomics Transistor for Electronic Detection of SARS‐Cov2 Variants Antigen and Viral RNA Without Amplification

AbstractThe SARS‐CoV‐2 pandemic caused a public health crisis throughout the world and highlighted the need for rapid and sensitive testing as a countermeasure. A sensitive and specific biosensor platform is developed for the detection of antigen and RNA of SARS‐CoV‐2, and its variant (B1.1.529). The demonstrated biosensor platform combines unique protein catalyzed capture bioreceptors (PCCs) for antigen capture and a chimeric (RNA‐DNA) probe for RNA detection using LwaCas13a collateral cleavage activity atop graphene field effect transistors (gFETs). The reported biosensor is able to differentiate unprocessed 104 pfu m−1 samples of SARS‐CoV‐2 from Influenza and Rhinovirus. The limit of detection (LOD) calculated for SARS‐CoV‐2 antigen is 103 in buffer and 104 PFU mL−1 in 10% saliva, while LOD of ≈65 am calculated for viral RNA isolate without amplification. To provide a high reliability of detection, the role of internal and external factors with respect to gate voltage is further analyzed by Principal Component Analysis (PCA). Based on PCA analysis, the authors are able to classify the samples as pathogen positive or negative (Y > 0: Positive for pathogen, Y < 0: Negative for pathogen). The reported platform can be quickly adapted for multi‐omics and multiplexed diagnosis of continuously evolving biothreats and global pandemics.

Abstract 2238: A novel comprehensive breakpoint-targeted assay for clinically actionable RNA fusions and aberrant RNAs in solid tumors

Abstract Background: Gene fusions are major drivers of cancer and their accurate detection is key for supporting diagnosis and therapy selection. There are more than 65,000 annotated gene fusion events. Current fusions detection strategies are limited to amplicon panels querying only a small number of genes, or whole exome capture based assays, which can reduce sensitivity. With these technologies, multiple small panels are required to capture all relevant fusions, which is prohibitive for small biopsies like fine needle aspirates. Hence, clinical grade assays able to accurately detect a comprehensive set of the most clinically relevant gene fusions for solid tumors are needed. Methods: We isolated total nucleic acid (TNA) from 141 FFPE tumor specimens or a control sample (Seraseq) and performed pair-ended, strand-specific hybridization-based RNA sequencing on a Next-Seq 500. We selected 252 fusion genes from NCCN and WHO guidelines, published clinical studies, and the 120 most frequent curated fusions in solid tumors from COSMIC (v91). These fusions are clinically relevant to most frequent cancers, including breast, colorectal, lung, lymphoma, pancreatic, prostate, salivary gland, sarcomas, and thyroid cancers. Chimeric probes were synthetized targeting fusion RNA sequences for 2230 selected breakpoints and exon junction regions of aberrant RNAs, including EGFRvIII, MET exon 14 skipping, ARv7 and ARv9. We also targeted the full coding region of 27 genes whose change in expression may suggest translocations and mutations, or have diagnostic value. Fusions were called by a custom pipeline using 3 fusion callers and a machine-learning algorithm. The PCR-based Archer FusionPlex assays and RT-PCR followed by Sanger were used as orthogonal validation methods. Results: This single RNA fusion assay can detect at least 1194 unique known fusions pairs involving 1104 genes, as compared to 63 or 47 fusion genes from other commercially available NGS assays. On 141 FFPE samples from various tumor types, we detected 100/106 fusions reported in our CLIA laboratory Archer Sarcoma and Comprehensive Thyroid & Lung (CTL) panels. We also identified 41 additional high confidence fusions missed by Archer but confirmed by Sanger sequencing. Thus, the assay has 95.9% (141/147) sensitivity and 100% specificity, as all fusions were confirmed by either orthogonal assay. We also detected MET exon 14 skipping, EGFRvIII and ARv7 variants. Importantly, in two samples we detected MET exon 14 skipping not predicted from DNA mutation analysis showing the sensitivity of the approach. Notably, several of the 41 additional fusions are novel and were targeted by only one of the breakpoints. Conclusion: We developed a novel and efficient breakpoint targeted fusion detection RNA-seq assay from extracted TNA from FFPE samples that can comprehensively profile thousands of clinically actionable RNA fusions and aberrant RNAs in solid tumors. Citation Format: Fernando J. Lopez-Diaz, Steven P. Rivera, Chenyin Ou, Christophe Magnan, Brad Thomas, Tibor Gyuris, Yanglong Mou, Segun Jung, Madhuri Paul, Forrest Blocker, Shari Brown, Jacqueline Lekostaj, Ryan Bender, Sally Agersborg, Lawrence M. Weiss, Vincent Funari. A novel comprehensive breakpoint-targeted assay for clinically actionable RNA fusions and aberrant RNAs in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2238.

Abstract 241: An efficient probe design algorithm for direct fusion targeting from RNA

Abstract Background: The use of sequencing technologies to detect gene fusions (GFs) from RNA shows promising results for the future of cancer diagnosis and treatment. Major obstacles for this approach include target design and lack of well-curated databases of RNA breakpoints. Currently, off-the-shelf designs include full transcript targeting that results in massive and costly amounts of data. Directly targeting the known GFs from RNA by designing probes targeting the fusion junction sequence is studied here as an alternative to whole-exome sequencing (WES). We present notably a novel algorithm capable of designing the probes to accurately target the desired fusions from RNA. Methods: For a given GF detected either from DNA or from RNA, the algorithm is as follows: (1) Collect gene and isoform information for both partners from seven public databases; (2) For each candidate pair of isoforms, locate where the breakpoints will be observed and assign a score based on various criteria such as sequence completion, coding information, transcript support level, % identity with and % visible on hg38; (3) Select the top scoring pair of transcripts and extract the chimeric probe sequence. Two sets of probes extracted with this protocol targeting 524 and 1632 known GFs were synthetized and tested on several samples (Table 1). The Agilent SureSelect Human All Exon V6 capture kit was used to compare targeting efficiency against WES. Results: Targeted enrichment of a SeraSeq control showed a 5 to 20 fold increase in supporting evidence over WES. On 10 clinical samples, we observed 10-30x increase in supporting reads. A higher sensitivity is observed in both cases. Conclusion: We developed a novel algorithm capable of accurately identifying the most likely location of an RNA fusion junction and generating the probe sequences for oligo synthesis. This method not only enriches for more supporting data but also reduces the associated costs. Average number of supporting reads per fusion per million reads for WES & direct targetingSampleKnown FusionWES (A)WES (B)524 TF (A)524 TF (B)1632 TF (A)1632 TF (B)SeraSeq 0710-0496CCDC6→RET8102703558178CD74→ROS13581592832246250EGFR→SEPTIN1418172343159180FGFR3→BAIAP2L114542832612572FGFR3→TACC3239861879270203LMNA→NTRK123112152807167PAX8→PPARG29191932466662SLC34A2→ROS1102217642589142SLC45A3→BRAF1115433420141105TFG→NTRK13540275377128132TMPRSS2→ERG0055934817079TPM3→NTRK11523246359106117Avg. SeraSeq12 Fusions1821373430132116Clinical S1EML4→ALK2515344NA81NAClinical S2EWSR1→FLI15738514NA111NAClinical S3TES→MET2030115NA75NAClinical S4EZR→ROS14311,059NA266NAClinical S5SDC4→ROS1553,354NA1,082NAClinical S6SH3BP5→PPARG004NA1NAClinical S7H2BC21→NTRK11118NA4NAClinical S8COL1A1→PDGFB2092505,530NA2,289NAClinical S9KIF5B→RET2424437NA174NAClinical S10POC1B→GLI1158416NA161NAAvg. Clinical10 Fusions36401,179NA424NA Citation Format: Christophe N. Magnan, Steven P. Rivera, Fernando J. Lopez-Diaz, Chen-Yin Ou, Kenneth B. Thomas, Hyunjun Nam, Lawrence M. Weiss, Segun C. Jung, Vincent A. Funari. An efficient probe design algorithm for direct fusion targeting from RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 241.

Tissue microRNAs in non-small cell lung cancer detected with a new kind of liquid bead array detection system

BackgroundCommonly used miRNA detection methods cannot be applied for high-throughput analyses. However, this study was aimed to performed a liquid bead array detection system (LBAS) to detect tissue 6 miRNAs in non-small cell lung cancer (NSCLC).MethodsIn this study, evaluation of LBAS was performed to observe the precision, specificity, limitation and stability. Then, a total of 52 primary NSCLC patients who received resection operation without preoperative radiotherapy and chemotherapy between June 2013 and March 2014 were selected, and then the total RNA of the tissues were extracted. We prepared six NSCLC-related miRNAs for LBAS. After optimization and evaluation, LBAS was verified by detecting the relative expression levels of 6 microRNAs in the pathological tissues and corresponding normal tissues of 52 NSCLC patients.ResultsThe results of evaluation of LBAS showed that the Mean Fluorescence Intensity (MFI) of the reaction only added with chimeric probes and beads showed no significant change after 180 days (P > 0.05). And the intra-assay Coefficient of Variation (CV) was between 1.57 and 3.5%, while the inter-assay CV was between 4.24 and 11.27%, indicating this system was ideal for diagnostic reagents. In addition, only the beads corresponding to the additional miRNAs showed high MFIs from 8426 to 18,769, whereas the fluorescence values of the other beads were under background levels (MFIs = 20 to 55) in each reaction, indicating no cross reactivity among the miRNAs. The limit of detection of miR-21, miR-210, miR-125b, miR-155, miR-375, and miR-31 were 5.27, 1.39, 1.85, 2.01, 1.34, and 2.73 amol/μL, respectively, showing that the lowest detection limit of miRNA by this system was under pM level. Then, the relative expression levels of miR-21, miR-210, miR-125b, miR-155, miR-375, and miR-31 by using this system were significantly correlated with NSCLC (P < 0.05). And the results of AUC method indicated that specific of the LBAS system was 94.2%.ConclusionsOur findings suggest that LBAS was simple, high-throughput, and freely combined with absolute quantification. Thus, this system could be applied for tumor miRNAs detection.

Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection

Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1 DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3′-OH RNA/5′-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5′ flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide polymorphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.

Sequential or multiplex electrochemical detection of miRs based on the p19 function relative to three sandwiches of different structural hybrids on the liposomal sensor

In this work, we designed a liposomal electrochemical sensor with DOTAP-DOPE liposome, chimeric probes, p19 as a caliper molecule, and the competitor structural hybrid (just RNA) for detection of three micro-RNAs in one SPCE/GNP electrode. The sensor is stabled when the cationic spherical DOTAP-DOPE liposomes sandwich with hybrids of the different sandwiched of probes (T-M-linear, Stem) and 21-124a-221miRs. With the addition of P19, in the presence of a sandwiched competitor (T-linear/21miR), the system is stable (ON) and is shut off in the presence of structural sandwiched hydrides of M-linear+124a/Stem+221 miR due to the lack of adequate access to segments of RNA-miRs of chimeric probes. For the first time in this study, three probes were sandwiched on the separate liposome for sequential identification of 21-124a-221 or multiplex detection of miRs (221 or 124a with 21) with high specificity and sensitivity (as low as 0.1 fM). Electrochemical impedance (EIS) were performed for sensing three miRs in PBS containing 1 mM [Fe(CN)6]−3/−4 which DOTAP-DOPE liposome acted as an enhancing intermediate layer in the electrochemical reactions. Transmission Electron Microscopy (TEM)), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Ultraviolet–Visible (UV) spectroscopic techniques are used to understand the interactions between the DOPE-DOTAP, AuNP, different probes, miRs and p19.

RETRACTED ARTICLE: Electrochemical Sensor for Detection of miRs Based on the Differential Effect of Competitive Structures in The p19 Function

The present study aim to design a liposomal electrochemical sensor using 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and dioleoylphosphatidylethanolamine(DOPE), chimeric probes and p19, it has been considered as a caliper molecule as well. Also the competitor structural hybrid (RNA) was used to detect three types of miRs in one screen printed electrode modified by gold nanoparticle (SCPE/GNP). In this purpose, the sensor signal stabilized when the cationic DOTAP-DOPE with hybrids of the chimeric probes (Stem, M-linear) sandwiched in order to detect 221–124a miRs. Given the lack of accessibility to RNA-miRs segments of chimeric probes, p19 inhibited the electrochemical reaction and shifted signal to off. After that p19 connected with the free hybrid of T-linear/21miR (just RNA) as competing for structure and the signal was shifted to ON, again. In this study, the electrochemical measurements were performed between the potentials at −0.4 V and +0.4 V with 1 mM [Fe(CN)6]-3-/4 which DOTAP-DOPE acted as an enhancer layer in the electrostatically reaction. This sensor determines as low as 0.4 fM of miRNA with high selectivity and specificity for sequential analysis of 124a-221-21 miRs in just 2 h.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

Neutralized chimeric DNA probe for detection of single nucleotide polymorphism on surface plasmon resonance biosensor

An implementation of neutralized chimeric DNA oligomer as a probe for sensitive detection of single nucleotide polymorphisms (SNPs) on a surface plasmon resonance imaging sensor is investigated. The chimeric DNA oligomer was synthesized in a conventional DNA synthesizer, containing neutral nucleotides with a methylated phosphate group. The secondary structures and melting points of the chimeric DNA fragment and its complexes with perfect-matched and single-mismatched complementary DNA molecules were examined by using circular dichroism and UV–vis spectroscopy in comparison with the native probe DNA counterpart. The results indicate that the chimeric DNA complexes can form a B-form structure and exhibit high thermostability. Moreover, the hybridization and discrimination efficiency of the chimeric probe DNA for the SNP genotyping were verified by using the SPRi sensor under different experimental conditions. The data reveal the effects of the ionic strength and operation temperature on the selectivity of the chimeric probe DNA for the SNP detection. The hybridization condition with a low ionic strength and high temperature allows the chimeric probe DNA distinguishing perfect-matched and single-mismatched target DNA molecules to the best extent, likely due to the reduced electrostatic repulsive force and presence of the additional methyl group on the backbone. Consequently, the direct and label-free detection with the SPR technique and neutralized chimeric probe DNA can be realized for the SNP genotyping by optimizing the operation condition and sequence design.

Near-infrared bioluminescent proteins for two-color multimodal imaging.

Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 104 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification.

Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis.

Phagocytosis is a receptor-mediated process whereby professional phagocytes internalize invading pathogens and apoptotic bodies into an intracellular vacuole or phagosome, leading to their degradation. During the formation and maturation of the phagosome, several lipids undergo changes and effector proteins are recruited on the nascent phagosome in a concerted manner. These highly localized, dynamic, and transient processes can only be studied by methods capable of high spatial and temporal resolution. The use of genetically encoded chimeric constructs coupled with fluorescence confocal microscopy enables the continuous, noninvasive analysis of the distribution and metabolism of lipids and effector proteins during phagocytosis. Here, we describe a method where the mouse macrophage cell line, RAW 264.7, and primary macrophages are transiently transfected with fluorescent chimeric probes to analyze and quantify phagocytosis of immunoglobulin-opsonized particles, using confocal microscopy.

A target-triggered dual amplification strategy for sensitive detection of microRNA

The accurate and quantitative analysis of microRNA (miRNA) expression is critical for biomedical research and clinical theranostics. In this study, we report a novel sensor for the sensitive detection of miRNA based on a duplex-specific nuclease (DSN)-assisted dual signal amplification strategy. A chimeric probe (DNA/2-OMe-RNA) that consists of a miRNA recognition DNA sequence and a Taqman probe hybridization RNA sequence (2′-O-methyl RNA) was designed and synthesized. One molecule of target miRNA can trigger cyclical cleavage of the chimeric probes to produce 2′-O-methyl RNA by DSN in the first round of amplification. The 2′-O-methyl RNA molecules can subsequently hybridize with Taqman probes and initiate the second round of cyclical amplification to generate detectable fluorescence by DSN. The proposed strategy exhibits high specificity in discriminating cognate miRNAs, and the dual signal transduction process enables the detection of miRNA concentrations as low as 7.3fM. We further applied this assay to miRNA quantification in cancer cells to confirm its applicability. The present study provides a sensitive, specific and simple method for miRNA detection and holds great potential for further application in biomedical research and in the clinical laboratory.

Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.

The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and—more recently—engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probes are chimeras between pseudocomplementary DNA (pcDNA) and Invader probes, which are activated for mixed-sequence dsDNA-recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalized nucleotides, respectively. We demonstrate that certain pseudocomplementary Invader probe designs result in very efficient and specific recognition of model dsDNA targets in buffers of high ionic strength. These chimeric probes, therefore, present themselves as a promising strategy for mixed-sequence recognition of dsDNA targets for applications in molecular biology and nucleic acid diagnostics.

Cycling probe technology to quantify and discriminate between wild-type varicella-zoster virus and Oka vaccine strains

Rapid differentiation between wild-type varicella zoster virus (VZV) and Oka-vaccine (vOka) strains is important for monitoring side reactions of varicella vaccination. To develop a high-throughput molecular diagnostic method for the differentiation of wild-type VZV and vOka strains based on cycling probe technology. The primers were designed to amplify common sequences spanning a single nucleotide polymorphism (SNP) in gene 62 of VZV. DNA–RNA chimeric probes (cycling probes) were designed to detect the SNP at nucleotide 105705. The cycling probe real-time PCR assays for VZV wild-type and vOka strains specifically amplified plasmids containing target sequences that ranged between 10 and 1×106 copies per reaction. The inter- and intra-assay coefficients of variation were less than 5%. After initial validation studies, the clinical reliability of this method was evaluated using 38 swab samples that were collected from patients suspected of being zoster. Compared to the loop mediated isothermal amplification method, which is defined as the gold standard, cycling probe real-time PCR was highly sensitive and specific. The cycling probe real-time PCR technology is a reliable tool for differentiating between wild-type VZV and vOka strains in clinical samples.

MiR-21 expression in the tumor cell compartment holds unfavorable prognostic value in gliomas

High-grade gliomas are some of the most lethal forms of human cancer, and new clinical biomarkers and therapeutic targets are highly needed. MicroRNAs (miRNAs), a group of short noncoding RNAs, hold great potential as new biomarkers and targets as they are commonly deregulated in a variety of diseases including gliomas. MicroRNA-21 (miR-21) is the most consistently overexpressed miRNA in several cancers including gliomas and is therefore very promising as a useful clinical biomarker and therapeutic target. To better understand the role of miR-21 in gliomas, paraffin-embedded glioma tissue samples from 193 patients with gradeI, II, III, and IV tumors were analyzed by insitu hybridization (ISH) using LNA-DNA chimeric probes. We found miR-21 expression in tumor cells and tumor-associated blood vessels, whereas no expression was seen in adjacent normal brain parenchyma. Using advanced image analysis we obtained quantitative estimates reflecting the miR-21 expression levels in each of these compartments. The miR-21 levels correlated significantly with grade [p=0.027, r (s)=0.161, 95% confidence interval (CI), 0.015-0.301] with the highest levels measured in glioblastomas. Only tumor cell miR-21 was associated with poor prognosis when adjusting for known clinical parameters (age, grade, and sex) in a multivariate analysis [p=0.049, hazard ratio (HR)=1.545, 95% CI, 1.002-2.381]. In conclusion, we have shown that miR-21 is located in both tumor cells and tumor blood vessels and that its level in the tumor cell compartment holds unfavorable prognostic value in gliomas.

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH).

Tiny Molecular Beacons: LNA/2′-O-methyl RNA Chimeric Probes for Imaging Dynamic mRNA Processes in Living Cells

New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2'-O-methyl RNA chimera oligonucleotides "tiny molecular beacons". We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons and the flexibility to couple them to a large variety of fluorophores and quenchers render them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for oskar mRNA and microinjected them into living Drosophila melanogaster oocytes. We then imaged the live oocytes via spinning disk confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons and are able to access previously inaccessible target regions.

Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides.

Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine.