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ABSTRACT: We synthesized a series of crosslinked photoresponsive polymeric particles with photolabile monomers and crosslinkers through miniemulsion polymerization. These particles are quite stable in dark, while light irradiation caused the breakage of particles and the efficient release of encapsulated contents up to 95 % based on Nile red fluorescence. Photoswitches of particle systems were confirmed by fluorescence spectroscopy, SEM and colorimetry. Particle uptake and triggered release in RAW264.7 cells were confirmed by fluorescein diacetate loaded particles. © 2013 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2013 The American Society of Photobiology.
Photochemistry and Photobiology 01/2013; · 2.41 Impact Factor
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ABSTRACT: A coumarin-based fluorescence chemoprobe was developed and evaluated for the selective and sensitive detection of hydrogen sulfide in degassed PBS buffers and fetal bovine serum. Fluorescence detection of hydrogen sulfide in living cells was also successfully achieved using two-photon confocal fluorescence imaging. Further in situ visualization of endogenous H(2)S was realized in cardiac tissues of normal rats and atherosclerosis (AS) rats.
The Analyst 12/2012; · 4.23 Impact Factor
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ABSTRACT: We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2'-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies.
Nucleic Acids Research 10/2012; · 8.03 Impact Factor
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ABSTRACT: A selective and sensitive fluorescence probe for hydrogen sulfide (H(2)S) detection was synthesized and evaluated in PBS buffer and fetal bovine serum. The effect of pH on the probe was also studied. In addition, visualization of H(2)S in Hela cells was achieved using confocal laser scanning fluorescence microscopy.
Analytical and Bioanalytical Chemistry 08/2012; 404(6-7):1919-23. · 3.78 Impact Factor
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ABSTRACT: We have used a photocaging strategy to develop novel phosphoramidites and expand the repertoire of protecting groups for modification of oligonucleotides by solid-phase synthesis. We synthesised five photolabile phosphoramidites and four new photolabile controlled pore glasses (CPGs). By using these photolabile phosphoramidites and CPGs, modified oligodeoxynucleotides (ODNs) with phosphate, amine, acid, thiol and carbonyl moieties at 5' and/or 3' ends were readily synthesised. To the best of our knowledge, this is the first report of introducing a carbonyl at the 5' end and thiol groups at both ends of ODNs with photolabile modifiers. Terminal labelling was also easily realised in solution or by on-column solid-phase synthesis. By using the photolabile amine modifier and the photolabile acid CPG, cyclisation of an oligodeoxynucleotide was achieved with good yields. This study provides an alternative way to introduce functional groups into oligonucleotides and expand the scope of oligonucleotide bio-orthogonal labelling.
Chemistry 07/2012; 18(31):9628-37. · 5.93 Impact Factor
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ABSTRACT: Light-responsive drug delivery systems are particularly appealing that are capable of releasing active molecules at the appropriate site and rate. We synthesized a series of photodegradable polymers that can form nanoparticles for drug encapsulation. These particles in aqueous solutions are stable in buffers with different pHs or at evaluated temperatures, while light can trigger the crash of particles and the release of encapsulated substances. The release efficiency can reach up to 90% based on Nile red fluorescence intensity upon 15 min light irradiation. Nanoparticle uptake by phagocytic cells and light-triggered release in cells were observed by fluorescence emission of the hydrolyzed fluorescein diacetate upon photoinduced degradation of these nanoparticles. No significant toxicity of these nanoparticles was found at the concentrations up to 1000 μg/mL before or after light irradiation. Further encapsulation and triggered release of a bioactive model drug (Tagalsin G) was evaluated for RAW 264.7 cells. Tagalsin G encapsulated in nanoparticles did not show cytotoxity to cells, while light triggered the release of Tagalsin G increasing cell death dramatically from 9% to 67%. Our model studies show a new promising strategy to trigger drug release in cells.
Langmuir 05/2012; 28(25):9387-94. · 4.19 Impact Factor
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ABSTRACT: Photodegradable polyesters were synthesized with a photolabile monomer 2-nitrophenylethylene glycol and dioyl chlorides with different lengths. These polymers can be assembled to form polymeric particles with encapsulation of target substances. Light activation can degrade these particles and release payloads in both aqueous solutions and RAW 264.7 cells.
International Journal of Molecular Sciences 01/2012; 13(12):16387-99. · 2.60 Impact Factor
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ABSTRACT: Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson-Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics.
ChemBioChem 11/2011; 12(18):2863-70. · 3.94 Impact Factor
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ABSTRACT: We intend to form photosensitive block copolymer micelles for controllable release of encapsulated substances. Here, we designed and synthesized a new photocleavable cross-linker (2-nitrophenyl ethylene glycol dimethacrylate) for methyl methacrylate (MMA) atom transfer radical polymerization. Four different ratios (0:1, 1:26, 1:16, 1:8.8) of the photocleavable cross-linker to MMA monomer were used and four block copolymers (P0, P1, P2, P3) were synthesized with PEO-Br as the macroinitiator. Gel permeation chromatography and (1) H NMR studies showed that linear polymer molecules could be cross-linked by the photocleavable linker. The fluorescence studies of the encapsulated Nile Red (NR) showed that there were lower critical micelle concentrations for the polymer P1, P2 and P3 than polymer P0. And dynamic light scattering and SEM confirmed the formation of polymer micelles. Photolysis experiments demonstrated that NR encapsulated in the polymer micelles could be released upon UV irradiation (365 nm, 11 mW cm(-2)) due to the breakage of the photocleavable linker and the generation of more hydrophilic acid moieties, which destabilized polymer micelles. Our study shows a new strategy for the possibility of photocontrollable drug release for hydrophobic drugs.
Photochemistry and Photobiology 01/2011; 87(3):646-52. · 2.41 Impact Factor
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ABSTRACT: A dumbbell molecular beacon (DMB) was designed and synthesized with the attachment of a fluorophore and a quencher at two ends. This DMB probe can be used to detect single mismatch of a 20mer oligodeoxynucleotide in two different buffers and discrimination factors were as high as 60 at 37°C. Statistics of single substitutions of analytes showed that both substituted positions and substituted nucleotides have important contributions for this probe to efficiently distinguish the true analyte from mismatched ones. Hybridization kinetics of DMB with the target oligonucleotide was also studied.
Bioorganic & medicinal chemistry letters 09/2010; 20(22):6547-50. · 2.65 Impact Factor
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ABSTRACT: Caged antisense oligodeoxynucleotides (asODNs) are synthesized by linking two ends of linear oligodeoxynucleotides using a photocleavable linker. Two of them (H30 and H40) have hairpin-like structures which show a large difference in thermal stability (Delta T(m) = 17.5 degrees C and 11.6 degrees C) comparing to uncaged ones. The other three (C20, C30 and C40) without stable secondary structures have the middle 20 deoxynucleotides complementary to 40-mer RNA. All caged asODNs have restricted opening which provides control over RNA/asODN interaction. RNase H assay results showed that 40-mer RNA digestion could be photo-modulated 2- to 3-fold upon light-activation with H30, H40, C30 and C40, while with C20, RNA digestion was almost not detectable; however, photo-activation triggered >20-fold increase of RNA digestion. And gel shift assays showed that it needed >0.04 microM H40 and 0.5 microM H30 to completely bind 0.02 microM 40-mer RNA, and for C40 and C30, it needed >0.2 microM and 0.5 microM for 0.02 microM 40-mer RNA binding. However, even 4 microM C20 was not able to fully bind the same concentration of 40-mer RNA. By simple adjustment of ring size of caged asODNs, we could successfully photoregulate their hybridization with mRNA and target RNA hydrolysis by RNase H with light activation.
Nucleic Acids Research 02/2010; 38(11):3848-55. · 8.03 Impact Factor
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ABSTRACT: 'RNA bandages' are composed of two 6-12-mer 2'-OMe RNA strands complementary to a mRNA target that are joined by a photocleavable linker. These tandem oligonucleotides typically exhibit much higher affinity for the mRNA than the individual strands. An RNA bandage with binding arms of different lengths and a 4-base gap blocked translation in vitro of GFP mRNA; subsequent near-UV irradiation restored translation. This provides a general method of photomodulating hybridization for a variety of oligonucleotide-based technologies.
Bioorganic & medicinal chemistry letters 10/2008; 18(23):6255-8. · 2.65 Impact Factor
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ABSTRACT: Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the 'caged' state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (T(m)) upon UV irradiation (DeltaT(m) = -29 degrees C). The most thermally stable conjugate, C6 (T(m) = 84 degrees C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex.
Nucleic Acids Research 03/2008; 36(2):559-69. · 8.03 Impact Factor
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Journal of the American Chemical Society 10/2007; 129(36):11000-1. · 9.91 Impact Factor
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ABSTRACT: The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.
BioTechniques 09/2007; 43(2):161, 163, 165 passim. · 2.67 Impact Factor
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Dalton Transactions 03/2007; · 3.84 Impact Factor
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ABSTRACT: Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.
Molecular BioSystems 03/2007; 3(2):100-10. · 3.53 Impact Factor
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Angewandte Chemie International Edition 06/2006; 45(21):3523-6. · 13.45 Impact Factor
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ABSTRACT: The activity of a 20-mer antisense oligodeoxynucleotide (asODN) is transiently blocked by attaching a partially complementary sense strand (sODN) via a heterobifunctional photocleavable linker (PL). The asODN-PL-sODN conjugate forms a DNA hairpin-like structure that is considerably more stable than the corresponding asODN/sODN duplex. In conjugate form, the asODN is prevented from hybridizing to exogenous RNA or DNA molecules. Activity is restored after modest exposure to UV light (lambda approximately 365 nm). Here, we provide a detailed procedure for synthesizing photoactive asODNs in good yields. Synthesis, purification and analysis of the light-activated asODN can be completed within 1-2 weeks.
Nature Protocol 02/2006; 1(6):3041-8. · 8.36 Impact Factor
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ABSTRACT: The DNA polymerase reaction by Klenow fragment (KF) was efficiently regulated with UV light using a 25-mer caged fluorescent oligodeoxynucleotide (CFO) as the template. The CFO was functionalized with a fluorescein reporter (Fl) and photocleavable DABSYL quencher moiety (Dab). With Fl and Dab at adjacent cytidines in the middle at the template, KF was blocked from extending a complementary 12-mer primer. Upon UV photolysis of the DABSYL blocking group under aerobic conditions, fluorescein emission was restored and 50% of the primers were fully extended by KF.
Bioorganic & Medicinal Chemistry Letters 01/2006; 15(23):5303-6. · 2.55 Impact Factor
