Superior structure stability and selectivity of hairpin nucleic acid probes with an l-DNA stem

Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2′-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications

An Allosteric-Probe for Detection of Alkaline Phosphatase Activity and Its Application in Immunoassay

A fluorescence strategy for alkaline phosphatase (ALP) assay in complicated samples with high sensitivity and strong stability is developed based on an allosteric probe (AP). This probe consists of two DNA strands, a streptavidin (SA) aptamer labeled by fluorophore and its totally complementary DNA (cDNA) with a phosphate group on the 5′ end. Upon ALP introduction, the phosphate group on the cDNA is hydrolyzed, leaving the unhydrolyzed cDNA sequence for lambda exonuclease (λ exo) digestion and releasing SA aptamer for binding to SA beads, which results in fluorescence enhancement of SA beads that can be detected by flow cytometry or microscopy. We have achieved a detection limit of 0.012 U/mL with a detection range of 0.02~0.15 U/mL in buffer and human serum. These figures of merit are better than or comparable to those of other methods. Because the fluorescence signal is localized on the beads, they can be separated to remove fluorescence background from complicated biological systems. Notably, the new strategy not only applies to ALP detection with simple design, easy operation, high sensitivity, and good compatibility in complex solution, but also can be utilized in ALP-linked immunosorbent assays for the detection of a wide range of targets

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies

Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR

An agarose droplet method was developed for highly parallel and efficient single molecule emulsion PCR. The method capitalizes on the unique thermoresponsive sol-gel switching property of agarose for highly efficient DNA amplification and amplicon trapping. Uniform agarose solution droplets generated via a microfluidic chip serve as robust and inert nanolitre PCR reactors for single copy DNA molecule amplification. After PCR, agarose droplets are gelated to form agarose beads, trapping all amplicons in each reactor to maintain the monoclonality of each droplet. This method does not require cocapsulation of primer labeled microbeads, allows high throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.National Scientific Foundation of China [20805038, 21075104, 20620130427]; National Basic Research Program of China [2007CB935603, 2010CB732402

A T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection

National Basic Research Program of China [2010CB732402, 2013CB933703]; National Science Foundation of China [21205100, 21275122, 21075104]; National Instrumentation Program [2011YQ03012412]; Fundamental Research Funds for the Central Universities [2012121025]; Natural Science Foundation of Fujian Province for Distinguished Young Scholars [2010J06004]A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA-CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity

Backbone-modified molecular beacons for highly sensitive and selective detection of microRNAs based on duplex specific nuclease signal amplification

National Basic Research Program of China [2010CB732402, 2013CB933703]; National Science Foundation of China [21205100, 21275122]; National Instrumentation Program [2011YQ03012412]; Natural Science Foundation of Fujian Province for Distinguished Young Scholars [2010J06004]Based on backbone-modified molecular beacons and duplex-specific nuclease, we have developed a target recycling amplification method for highly sensitive and selective miRNA detection. The combination of a low fluorescence background of 2-OMe-RNA modified MB and nuclease-assisted signal amplification leads to ultrahigh assay sensitivity, and the powerful discriminating ability of MB enables the differentiation of highly similar miRNAs with one-base difference, both of which are of great significance to miRNA detection

Metabolic Labeling of Peptidoglycan with NIR-II Dye Enables in vivo Imaging of Gut Microbiota.

Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real-time and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near-infrared (NIR-II)-based method for in vivo imaging of gut bacteria. Using D-propargylglycine in gavage and then click reaction with an azide-containing NIR-II dye, gut microbiota of a donor mouse was strongly labeled with NIR-II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. We then adopted this chemical strategy to image different bacterial species, which expanded its applicability in microbiology. Moreover, by employing this method, we found that the biogeography of gut microbiota was dramatically affected by host’s gastrointestinal motilities. The NIR-II-based metabolic labeling strategy reported here, to our knowledge, provides the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut "dark matters"

Single-molecule photon-fueled DNA nanoscissors for DNA cleavage based on the regulation of substrate binding affinity by azobenzene

National Basic Research Program of China [2010CB732402]; National Scientific Foundation of China [21205100, 21075104]; Natural Science Foundation of Fujian Province for Distinguished Young Scholars [2010 J06004]A pair of single-molecule photo-responsive DNA nanoscissors for DNA cleavage based on the regulation of substrate binding affinity was designed and fabricated. Compared with other DNA nanomachines, our DNA nanoscissors have the advantages of a clean switching mechanism, as well as robust and highly reversible operation

A diazirine-based photoaffinity probe for facile and efficient aptamer-protein covalent conjugation

National Basic Research Program of China [2010CB732402, 2013CB933703]; National Science Foundation of China [91313302, 21205100, 21275122, 21075104]; Fundamental Research Funds for the Central Universities [2012121025]; National Science Foundation for Distinguished Young Scholars of China [21325522]A photo-reactive functional labelling reagent, diazirine phosphoramidite, was designed and synthesized for easy and flexible sitespecific labelling of oligonucleotides with the diazirine moiety. The new reagent allows facile photo-crosslinking of oligonucleotide with its interacting partner for a variety of applications, including tertiary structure determination, molecular interaction study and biomarker discovery

Chameleon clothes for quantitative oxygen imaging

We fabricated a chameleon cloth which changed its vivid colours at different oxygen concentrations under ultraviolet excitation. Combined with a photographing technique, the chameleon cloth possesses the ability for real-time quantitative imaging of oxygen distribution, which could be used for quick location of dangerous oxygen-deficiencies and oxygen-free regions.National Basic Research Program of China[2010CB732402]; National Nature Scientific Foundation of China[20975085, 20735002]; NFFTBS[J1030415