Catalytic formation of luminescent lanthanide complexes using an entropy-driven DNA circuit

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

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Distinguishing L from M photopigment coding sequences by hybridization to novel locked nucleic acid (LNA) oligonucleotide probes

Visual Neuroscience ◽

10.1017/s0952523808080607 ◽

2008 ◽

Vol 25 (3) ◽

pp. 283-287

Author(s):

CHRISTINA PETTAN-BREWER ◽

LI FU ◽

SAMIR S. DEEB

Keyword(s):

Nucleic Acid ◽

Locked Nucleic Acid ◽

Macaca Nemestrina ◽

Oligonucleotide Probes ◽

Coding Sequences ◽

Cone Mosaic ◽

Target Nucleic Acid ◽

High Degree

Many attempts have been made over the years to distinguish human and primate L (long-wavelength sensitive) from M (middle-wavelength sensitive) cone photoreceptors using either immunohistochemistry or in situ hybridization. These attempts have been unsuccessful due to the very high degree of identity between the sequences of the L and M proteins and encoding mRNAs. The recent development of chemically modified oligonucleotide probes, referred to as locked nucleic acid (LNA) probes, has shown that they hybridize with much greater affinity and specificity to the target nucleic acid. This has greatly increased the potential for differentiating L from M cones by in situ hybridization. We have designed LNA oligonucleotide probes that are complementary to either the L or M coding sequences located in exon 5 of the Macaca nemestrina L and M pigment genes. We have shown that the LNA-M and LNA-L probes hybridize specifically to their respective target nucleic acid sequences in vitro. This result strongly suggests that these probes would be instrumental in rapidly distinguishing L from M cone in the entire retina, and in defining the cone mosaic during development and in adults.

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RNAblueprint: flexible multiple target nucleic acid sequence design

Bioinformatics ◽

10.1093/bioinformatics/btx263 ◽

2017 ◽

Vol 33 (18) ◽

pp. 2850-2858 ◽

Cited By ~ 8

Author(s):

Stefan Hammer ◽

Birgit Tschiatschek ◽

Christoph Flamm ◽

Ivo L Hofacker ◽

Sven Findeiß

Keyword(s):

Nucleic Acid ◽

Nucleic Acid Sequence ◽

Multiple Target ◽

Sequence Design ◽

Target Nucleic Acid

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Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

BioTechniques ◽

10.2144/05391rr01 ◽

2005 ◽

Vol 39 (1) ◽

pp. 89-96 ◽

Cited By ~ 33

Author(s):

Régis Peytavi ◽

Liu-ying Tang ◽

Frédéric R. Raymond ◽

Karel Boissinot ◽

Luc Bissonnette ◽

...

Keyword(s):

Nucleic Acid ◽

Dna Hybridization ◽

Capture Probe ◽

Target Nucleic Acid ◽

Hybridization Efficiency ◽

Microarray Dna

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Ultra–sensitive droplet digital PCR for detecting a low–prevalence somatic GNAQ mutation in Sturge–Weber syndrome

Scientific Reports ◽

10.1038/srep22985 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 29

Author(s):

Yuri Uchiyama ◽

Mitsuko Nakashima ◽

Satoshi Watanabe ◽

Masakazu Miyajima ◽

Masataka Taguri ◽

...

Keyword(s):

Detection Limit ◽

Nucleic Acid ◽

Somatic Mutation ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Blood Leukocytes ◽

Target Nucleic Acid ◽

Weber Syndrome ◽

Sturge Weber Syndrome ◽

Blood Leukocyte

Abstract Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA–ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge–Weber syndrome (SWS) using ddPCR and PNA–ddPCR. Importantly, among three patients previously found to be mutation negative by next–generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS.

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5601976 Method for detecting target nucleic acid in specimen

Biotechnology Advances ◽

10.1016/s0734-9750(97)87756-7 ◽

1997 ◽

Vol 15 (3-4) ◽

pp. 730

Keyword(s):

Nucleic Acid ◽

Target Nucleic Acid

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Three-Way Junction-Induced Isothermal Amplification with High Signal-to-Background Ratio for Detection of Pathogenic Bacteria

Sensors ◽

10.3390/s21124132 ◽

2021 ◽

Vol 21 (12) ◽

pp. 4132

Author(s):

Jung Ho Kim ◽

Seokjoon Kim ◽

Sung Hyun Hwang ◽

Tae Hwi Yoon ◽

Jung Soo Park ◽

...

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Pathogenic Bacteria ◽

High Selectivity ◽

Isothermal Amplification ◽

Rna Aptamers ◽

High Signal ◽

Fluorescence Signal ◽

Enhanced Fluorescence ◽

Target Nucleic Acid

The consumption of water and food contaminated by pathogens is a major cause of numerous diseases and deaths globally. To control pathogen contamination and reduce the risk of illness, a system is required that can quickly detect and monitor target pathogens. We developed a simple and reproducible strategy, termed three-way junction (3WJ)-induced transcription amplification, to detect target nucleic acids by rationally combining 3WJ-induced isothermal amplification with a light-up RNA aptamer. In principle, the presence of the target nucleic acid generates a large number of light-up RNA aptamers (Spinach aptamers) through strand displacement and transcription amplification for 2 h at 37 °C. The resulting Spinach RNA aptamers specifically bind to fluorogens such as 3,5-difluoro-4-hydroxybenzylidene imidazolinone and emit a highly enhanced fluorescence signal, which is clearly distinguished from the signal emitted in the absence of the target nucleic acid. With the proposed strategy, concentrations of target nucleic acids selected from the genome of Salmonellaenterica serovar Typhi (S. Typhi) were quantitatively determined with high selectivity. In addition, the practical applicability of the method was demonstrated by performing spike-and-recovery experiments with S. Typhi in human serum.

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Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.751408 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jian Zhang ◽

Hailong Lv ◽

Linxian Li ◽

Minjie Chen ◽

Dayong Gu ◽

...

Keyword(s):

Nucleic Acid ◽

Pathogen Detection ◽

Direct Detection ◽

Detection Sensitivity ◽

Molecular Diagnostic ◽

Target Nucleic Acid ◽

Precise Diagnosis ◽

Important Approach ◽

Pathogen Diagnosis ◽

Diagnostic Technologies

Molecular diagnostic (MDx) methods directly detect target nucleic acid sequences and are therefore an important approach for precise diagnosis of pathogen infection. In comparison with traditional MDx techniques such as PCR, the recently developed CRISPR-based diagnostic technologies, which employ the single-stranded nucleic acid trans-cleavage activities of either Cas12 or Cas13, show merits in both sensitivity and specificity and therefore have great potential in both pathogen detection and beyond. With more and more efforts in improving both the CRISPR trans-cleavage efficiencies and the signal detection sensitivities, CRISPR-based direct detection of target nucleic acids without preamplification can be a possibility. Here in this mini-review, we summarize recent research progresses of amplification-free CRISPR-Dx systems and explore the potential changes they will lead to pathogen diagnosis. In addition, discussion of the challenges for both detection sensitivity and cost of the amplification-free systems will also be covered.

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Fluorescent ribonucleoside analogues as probes for investigating RNA structure and function

Pure and Applied Chemistry ◽

10.1351/pac-con-10-09-16 ◽

2010 ◽

Vol 83 (1) ◽

pp. 213-232 ◽

Cited By ~ 19

Author(s):

Seergazhi G. Srivatsan ◽

Anupam A. Sawant

Keyword(s):

Nucleic Acid ◽

Conformational Changes ◽

Rna Structure ◽

Photophysical Properties ◽

Local Environment ◽

Spectroscopic Techniques ◽

Rna Structures ◽

Base Pairs ◽

Target Nucleic Acid ◽

And Function

Numerous biophysical tools based on fluorescence have been developed to advance the understanding of RNA–nucleic acid, RNA–protein, and RNA–small molecule inter-actions. In this regard, fluorescent ribonucleoside analogues that are sensitive to their local environment provide sensitive probes for investigating RNA structure, dynamics, and recognition. Most of these analogues closely resemble the native ribonucleosides with respect to their overall dimension and have the ability to form canonical Watson–Crick (WC) base pairs. Therefore, it is possible to place these probes near the point of interaction in a target nucleic acid with minimum structural perturbations and gain insight into the intricacies of conformational changes taking place in and around the interaction site. Here, we provide a concise background on the development and recent advances in the applications of base-modified fluorescent ribonucleoside analogue probes. We first present various base-modified fluorescent ribonucleoside analogues, their photophysical properties, and methods to incorporate these analogues into oligoribonucleotides. We then discuss the established spectroscopic techniques, which make use of the fluorescence properties of these emissive ribonucleoside analogues. Finally, we present the applications of base-modified fluorescent ribonucleoside analogues used as probes incorporated into oligoribonucleotides in investigating RNA structures and functions.

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