Xinjing Tang

Regulating gene expression with light-activated oligonucleotides.

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.

Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides.

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 (Tm) upon UV irradiation (ΔTm = −29°C). The most thermally stable conjugate, C6 (Tm = 84°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.

Photodegradable polyurethane self-assembled nanoparticles for photocontrollable release.

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.

Synthesis and Unique Photoluminescence Properties of Nitrogen‐Rich Quantum Dots and Their Applications

Nitrogen-rich quantum dots (N-dots) were serendipitously synthesized in methanol or aqueous solution at a reaction temperature as low as 50 °C. These N-dots have a small size (less than 10 nm) and contain a high percentage of the element nitrogen, and are thus a new member of quantum-dot family. These N-dots show unique and distinct photoluminescence properties with an increasing percentage of nitrogen compared to the neighboring carbon dots. The photoluminescence behavior was adjusted from blue to green simply through variation of the reaction temperature. Furthermore, the detailed mechanism of N-dot formation was also proposed with the trapped intermediate. These N-dots have also shown promising applications as fluorescent ink and biocompatible staining in C. elegans.

Taking control of gene expression with light-activated oligonucleotides

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.

Photophysical properties and TPA optical limiting of two new organic compounds

The emission and optical limiting properties of two new organic compounds have been experimentally investigated. Pumping by picosecond and nanosecond lasers, respectively, these two compounds possess strong two-photon absorption and optical limiting effects. The results of time-resolved fluorescence of them in DMF solvent at the different concentrations and small quantum yields indicate that the non-radiation process and reabsorption result in the low emission efficiency of these new molecules with large two-photon absorption (TPA) cross-sections.

Manipulation of gene expression in zebrafish using caged circular morpholino oligomers

Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their experimental applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.

Two-photon induced fluorescence of novel dyes

Abstract Three novel compounds, trans -2-[ p -( N -ethyl- N -(hydroxyethyl)amino)styryl]- N -methylbenzothiazolium iodide ( 1 ), trans -2-[ p -( N -ethyl- N -(hydroxyethyl)amino)styryl]-1′,3′,3′-trimethylindolium iodide ( 2 ), and trans -2-[ p -( N , N -dimethylamino)styryl]-1′,3′,3′-trimethylindolium iodide ( 3 ), were synthesized and their two-photon induced fluorescence behavior was studied. Under excitation by 1064 nm laser irradiation, the solutions of these compounds exhibit two-photon induced fluorescence with λ max at 639, 666 and 665 nm for 1 , 2 and 3 respectively.

Quaternary Ammonium Promoted Ultra Selective and Sensitive Fluorescence Detection of Fluoride Ion in Water and Living Cells

Highly selective and sensitive fluorescent probes with a quaternary ammonium moiety have been rationally designed and developed for fast and sensitive fluorescence detection of fluoride ion (F(-) from NaF, not TBAF) in aqueous solution and living cells. With the sequestration effect of quaternary ammonium, the detection time was less than 2 min and the detection limit of fluoride ion was as low as 0.57 ppm that is among the lowest detection limits in aqueous solutions of many fluoride fluorescence probes in the literature.

Chemoselective reduction-based fluorescence probe for detection of hydrogen sulfide in living cells.

A selective and sensitive fluorescence probe for hydrogen sulfide (H2S) 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 H2S in Hela cells was achieved using confocal laser scanning fluorescence microscopy.