ABSTRACT
In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 104 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions.
Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Spatial Analysis , Transcriptome/genetics , Animals , Brain/growth & development , Embryo, Mammalian , Feasibility Studies , Genetic Techniques , HeLa Cells , Humans , Male , Mice , NIH 3T3 Cells , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/radiation effects , Reverse Transcription/radiation effects , Transcriptome/radiation effects , Ultraviolet RaysABSTRACT
Mechanical force applied by ultrasound in solution leads to the dissociation of DNA metallo-base-pair interactions when these motifs are functionalized with oligodeoxynucleotide sequences of sufficient length. The annealing and force-induced denaturing process is followed by the attachment of distance-sensitive fluorescent probes and is found to be reversible.
Subject(s)
DNA/chemistry , Oligodeoxyribonucleotides/chemistry , Silver/chemistry , Base Pairing , DNA/genetics , DNA/radiation effects , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Nucleic Acid Hybridization/radiation effects , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/radiation effects , Ultrasonic WavesABSTRACT
We synthesized several DNA oligonucleotides containing one or several 2'-O-methyl-8-methyl guanosine (m8Gm) and demonstrated that these oligonucleotides not only stabilize the Z-DNA with a wide range of sequences under low salt conditions but also possess high thermal stability. Using artificial nucleobase-containing oligonucleotides, we studied the interaction of the Zα domain with Z-DNA. Furthermore, we showed that the m8Gm-contained oligonucleotides allow to study the photochemical reaction of Z-DNA.
Subject(s)
DNA, Z-Form/chemistry , Guanosine/analogs & derivatives , Guanosine/chemistry , Oligodeoxyribonucleotides/chemistry , DNA, Z-Form/chemical synthesis , DNA, Z-Form/metabolism , DNA, Z-Form/radiation effects , Green Fluorescent Proteins/metabolism , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Oligodeoxyribonucleotides/radiation effects , Oxidation-Reduction , Protein Binding , Transition Temperature , Ultraviolet RaysABSTRACT
Hydrogen-rich DNA dinucleotide cation radicals (dGG + 2H)+â¢, (dCG + 2H)+â¢, and (dGC + 2H)+⢠represent transient species comprising protonated and hydrogen atom adducted nucleobase rings that serve as models for proton and radical migrations in ionized DNA. These DNA cation radicals were generated in the gas phase by electron-transfer dissociation of dinucleotide dication-crown-ether complexes and characterized by UV-vis photodissociation action spectra, ab initio calculations of structures and relative energies, and time-dependent density functional theory calculations of UV-vis absorption spectra. Theoretical calculations indicate that (dGG + 2H)+⢠cation radicals formed by electron transfer underwent an exothermic conformational collapse that was accompanied by guanine ring stacking and facile internucleobase hydrogen atom transfer, forming 3'-guanine C-8-H radicals. In contrast, exothermic hydrogen transfer from the 5'-cytosine radical onto the guanine ring in (dCG + 2H)+⢠was kinetically hampered, resulting in the formation of a mixture of 5'-cytosine and 3'-guanine radicals. Conformational folding and nucleobase stacking were energetically unfavorable in (dGC + 2H)+⢠that retained its structure of a 3'-cytosine radical, as formed by one-electron reduction of the dication. Hydrogen-rich guanine (G + H)⢠and cytosine (C + H)⢠radicals were calculated to have vastly different basicities in water, as illustrated by the respective p Ka values of 20.0 and 4.6, which is pertinent to their different abilities to undergo proton-transfer reactions in solution.
Subject(s)
Dinucleoside Phosphates/chemistry , Free Radicals/chemistry , Oligodeoxyribonucleotides/chemistry , Crown Ethers/chemistry , Dinucleoside Phosphates/chemical synthesis , Dinucleoside Phosphates/radiation effects , Electrons , Free Radicals/chemical synthesis , Free Radicals/radiation effects , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/radiation effects , Photons , Protons , Spectrophotometry, UltravioletABSTRACT
Recent studies have evidenced that oxidatively damaged DNA, which potentially leads to carcinogenic mutations and aging, may result from the direct absorption of low-energy photons (>250 nm). Herein, the primary species, i.e., ejected electrons and base radicals associated with such damage in duplexes with an alternating guanine-cytosine sequence are quantified by nanosecond transient absorption spectroscopy. The one-photon ionization quantum yield at 266 nm is 1.2 × 10-3, which is similar to those reported previously for adenine-thymine duplexes. This means that the simple presence of guanine, the nucleobase with the lowest ionization potential, does not affect photo-ionization. The transient species detected after 3 µs are identified as deprotonated guanine radicals, which decay with a half-time of 2.5 ms. Spectral assignment is made with the help of quantum chemistry calculations (TD-DFT), which for the first time, provide reference absorption spectra for guanine radicals in duplexes. In addition, our computed spectra predict the changes in transient absorption expected for hole localization as well as deprotonation (to cytosine and bulk water) and hydration of the radical cation.
Subject(s)
Cytosine/radiation effects , Free Radicals/chemical synthesis , Guanine/radiation effects , Oligodeoxyribonucleotides/radiation effects , Cytosine/chemistry , DNA Damage , Electrons , Guanine/chemistry , Half-Life , Models, Chemical , Oligodeoxyribonucleotides/chemistry , Photons , Quantum Theory , Ultraviolet Rays , Water/chemistryABSTRACT
The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.
Subject(s)
Luminescent Agents/chemistry , Oligodeoxyribonucleotides/chemistry , Oxygen/analysis , A549 Cells , Animals , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Coordination Complexes/radiation effects , Fluorescent Dyes/chemistry , Humans , Hydrogen-Ion Concentration , Light , Luminescent Agents/chemical synthesis , Luminescent Agents/radiation effects , Mice, Inbred BALB C , Molecular Imaging , Nitroimidazoles/chemical synthesis , Nitroimidazoles/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/radiation effects , Oxazines/chemistry , Oxygen/chemistry , Pyrenes/chemical synthesis , Pyrenes/chemistry , Ruthenium , Tumor HypoxiaABSTRACT
The photophysics and photochemistry of DNA is of great importance due to the potential damage of the genetic code by UV light. Quantum mechanical studies have played a key role in interpretating the results of modern time-resolved pump-probe spectroscopy, and in elucidating the main photoactivated reactive paths. This review provides a concise, complete picture of the computational studies carried out, approximately, in the past decade. We start with an overview of the photophysics of the nucleobases in the gas phase and in solution. We discuss the proposed mechanisms for ultrafast decay to the ground state, that involve conical intersections, consider the role of triplet states, and analyze how the solvent modulates the photophysics. Then we move to larger systems, from dinucleotides to single- and double-stranded oligonucleotides. We focus on the possible role of charge transfer and delocalized or excitonic states in the photophysics of these systems and discuss the main photochemical paths. We finish with an outlook on the current challenges in the field and future directions of research.
Subject(s)
DNA/radiation effects , Models, Chemical , Oligodeoxyribonucleotides/radiation effects , Purines/radiation effects , Pyrimidines/radiation effects , Base Pairing , DNA/chemistry , Hydrogen Bonding , Oligodeoxyribonucleotides/chemistry , Photochemical Processes , Purines/chemistry , Pyrimidines/chemistry , Quantum Theory , Ultraviolet RaysABSTRACT
A lot has been learned about the physical and chemical transformations that originate from the absorption of light by DNA, and computational chemistry has played a critical role in revealing the mechanisms of how these transformations occur. Nucleic acids consist of chromophores interacting via π stacking and hydrogen bonding. The fate of these systems after they absorb light is determined by the interplay and competition between pathways involving one chromophore or interacting chromophores. This Perspective highlights the role of π stacking in photophysical and photochemical processes in oligonucleotides and reveals the importance of excimers and exciplexes. Special types of excimers/exciplexes, characterized as bonded excimers/exciplexes, are also found to be important.
Subject(s)
Oligodeoxyribonucleotides/radiation effects , 2-Aminopurine/chemistry , 2-Aminopurine/radiation effects , Fluorescent Dyes/chemistry , Fluorescent Dyes/radiation effects , Light , Models, Chemical , Molecular Structure , Oligodeoxyribonucleotides/chemistry , Photochemical ProcessesABSTRACT
We achieved a conformational change of oligodeoxynucleotides and the regulation of DNAzyme function by means of a radiolytic strand exchange reaction of disulfide bond. We designed a system in which the DNAzyme function of RNA cleavage was suppressed by the hybridization of an inhibitor strand that possessed disulfide bond with an active DNAzyme. Hypoxic X-irradiation led to the recovery of RNA cleavage because the strand exchange reaction at the disulfide bond in inhibitor strand resulted in a release of inhibitor strand. This strategy may be applicable to gene regulation by hypoxic X-irradiation.
Subject(s)
DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , Disulfides/chemistry , Hypoxia , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Humans , Nucleic Acid Conformation , Oxidation-Reduction , X-RaysABSTRACT
We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N',N'-tetramethylrhodamine (F*) with oligodeoxyribonucleotides and a 2'-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD4 and conjugates of oligonucleotides with N,N,N',N'-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the F* is oxidized forming fluorescent F. This reaction is >30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells.
Subject(s)
DNA/chemistry , DNA/radiation effects , Light , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Photochemical Processes , Rhodamines/analysis , Base Sequence , Color , Molecular Sequence Data , Molecular Structure , Rhodamines/chemistryABSTRACT
An oligodeoxynucleotide (ODN) containing a 2',2'-difluorinated analogue of a C4'-oxidized abasic site (C4'-OAS) was designed for the amine modification of biomolecules that interact with nucleic acids. In contrast to the parent C4'-OAS, which yielded amine-modified products accompanied by DNA strand scission, the ODN containing the difluoro C4'-OAS efficiently yielded products carrying ODNs. The amine modification proceeded without additional reagents being required and might be applicable to reactions in biological systems.
Subject(s)
Amines/chemistry , Hydrocarbons, Fluorinated/chemical synthesis , Oligodeoxyribonucleotides/chemical synthesis , DNA/chemistry , DNA Damage , Hydrocarbons, Fluorinated/chemistry , Hydrocarbons, Fluorinated/radiation effects , Molecular Structure , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Oxidation-ReductionABSTRACT
DNA decoys have been developed for the inhibition of transcriptional regulation of gene expression. However, the present methodology lacks the spatial and temporal control of gene expression that is commonly found in nature. Here, we report the application of photoremovable protecting groups on nucleobases of nuclear factor κB (NF-κB) DNA decoys to regulate NF-κB-driven transcription of secreted alkaline phosphatase using light as an external control element. The NF-κB family of proteins is comprised of important eukaryotic transcription factors that regulate a wide range of cellular processes and are involved in immune response, development, cellular growth, and cell death. Several diseases, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease, have been linked to constitutively active NF-κB. Through the direct incorporation of caging groups into an NF-κB decoy, we were able to disrupt DNA:DNA hybridization and inhibit the binding of the transcription factor to the DNA decoy until UV irradiation removed the caging groups and restored the activity of the oligonucleotide. Excellent light-switching behavior of transcriptional regulation was observed. This is the first example of a caged DNA decoy for the photochemical regulation of gene expression in mammalian cells and represents an important addition to the toolbox of light-controlled gene regulatory agents.
Subject(s)
Gene Expression Regulation/radiation effects , NF-kappa B/metabolism , Oligodeoxyribonucleotides/radiation effects , Alkaline Phosphatase/genetics , Light , Methods , Photochemical Processes , Transcription, Genetic/drug effectsABSTRACT
Template-directed ligation of oligonucleotides by photochemical reaction has attracted much interest because of its biomedical and synthetic applications. In this study, we developed photoligaton of DNA by using a photoremovable protecting group and thiol-disulfide exchange reaction. A phosphoroamidite of o-nitrobenzyl derivatives were synthesized, and DNA modified with a nitrobenzyl-protected thiol group and disulfide group was synthesized by conventional phosphoroamidite chemistry using a DNA synthesizer. It was shown that photochemical reaction of a nitrobenzyl group with UV irradiation produced a free thiol group, leading to the DNA ligation through the thiol-disulfide exchange reaction.
Subject(s)
Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , DNA/chemistry , DNA/radiation effects , Disulfides/chemistry , Photochemical Processes , Sulfhydryl Compounds/chemistry , Ultraviolet RaysABSTRACT
The C4'-oxidized abasic site (1) is one of the oxidatively damaged DNA lesions induced by antitumor bleomycins. The reactivity of 1 with amine under neutral conditions giving lactam 2 and its structural similarity to unmodified DNA suggested the possibility that ODN containing 1 could react with proteins which interact with DNA at lysine residue. In order to provide nucleic acid analogues with useful lysine modifying reactivity, we planned to study reaction of 2'-substituted analogues of 1, such as 3-5, with amine and their corresponding caged precursors 6-8 were synthesized. Uncaging reaction of 16 containing 6 proceeded efficiently. The reaction of obtained 17 containing 3 with Z-Lys-OH did not afford products corresponding to lactam. However, HPLC analysis of the reaction mixture suggested interaction of 3 with Z-Lys-OH.
Subject(s)
DNA Damage , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/radiation effects , Oxidation-Reduction , Photochemical ProcessesABSTRACT
We describe a novel reversible interstrand photocrosslinking reaction of RNA. In this system, a modified oligodeoxynucleotide (ODN) containing 3-cyanovinylcarbazole nucleoside ((CNV)K) reacts by photoirradiation at 366 nm for 1 s with pyrimidine residue of a complementary template RNA to yield a crosslinked product in 94% yield.
Subject(s)
RNA/chemistry , Carbazoles/chemistry , DNA/chemistry , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Photochemical Processes , RNA/radiation effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The (6-4) photoproduct, which is one of the major UV-induced DNA lesions, causes carcinogenesis with high frequency. The (6-4) photolyase is a flavoprotein that can restore this lesion to the original bases, but its repair mechanism has not been elucidated. In this study, we focused on the interaction between the enzyme and the 3' pyrimidone component of the (6-4) photoproduct and prepared a substrate analogue in which the carbonyl group, a hydrogen-bond acceptor, was replaced with an imine, a hydrogen-bond donor, to investigate the involvement of this carbonyl group in the (6-4) photolyase reaction. UV irradiation of oligodeoxyribonucleotides containing a single thymine-5-methylisocytosine site yielded products with absorption bands at wavelengths longer than 300 nm, similar to those obtained from the conversion of the TT site to the (6-4) photoproduct. Nuclease digestion, MALDI-TOF mass spectrometry, and the instability of the products indicated the formation of the 2-iminopyrimidine-type photoproduct. Analyses of the reaction and the binding of the (6-4) photolyase using these oligonucleotides revealed that this imine analogue of the (6-4) photoproduct was not repaired by the (6-4) photolyase, although the enzyme bound to the oligonucleotide with considerable affinity. These results indicate that the carbonyl group of the 3' pyrimidone ring plays an important role in the (6-4) photolyase reaction. On the basis of these results, we discuss the repair mechanism.
Subject(s)
Deoxyribodipyrimidine Photo-Lyase/chemistry , Photochemical Processes , Animals , Chromatography, High Pressure Liquid , DNA Repair/genetics , DNA Repair/radiation effects , Deoxyribodipyrimidine Photo-Lyase/antagonists & inhibitors , Deoxyribodipyrimidine Photo-Lyase/radiation effects , Electrophoretic Mobility Shift Assay , Hydrogen Bonding/radiation effects , Hydrolysis/radiation effects , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/radiation effects , Photochemical Processes/radiation effects , Pyrimidinones/chemistry , Pyrimidinones/radiation effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity/genetics , Substrate Specificity/radiation effects , Ultraviolet Rays , Xenopus laevisABSTRACT
We report the nonenzymatic detection of 5-methylcytosine by using template-directed photoligation through 5-cyanovinyl-2'-deoxyuridine ((C)U) with high selectivity and present a new methylation detection method using a photoligation-based DNA chip assay.
Subject(s)
5-Methylcytosine/analysis , Oligonucleotide Array Sequence Analysis/methods , 5-Methylcytosine/chemistry , DNA/chemistry , DNA/radiation effects , DNA Probes/chemistry , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , PhotochemistryABSTRACT
DNA is usually found in nature as a double stranded helix but there are many other structural forms that it can adopt. Triple helical DNA structures are one of these arrangements that DNA can form through some folding interactions. These structures have attracted great interest in the field of DNA recognition due to their higher affinity and selectivity. Here we introduced a bimolecular triplex structure formed by a anthracene-DNA conjugate through a well characterized photodimerization reaction in the presence of complementary target and evaluated the system for the target discrimination by using a mismatch target. Results indicated that the system could provide moderate discrimination.
Subject(s)
Anthracenes/chemistry , DNA, Circular/chemistry , DNA/chemistry , Oligodeoxyribonucleotides/chemistry , Anthracenes/radiation effects , Base Pairing , Oligodeoxyribonucleotides/radiation effectsABSTRACT
In this paper, the synthesis of various vinyl substituted photosensitive pyrimidine nucleosides and nucleotides is described; starting from 5-Iodo-2'-deoxyuridine (IdU) or oligodeoxynucleotides (ODN) containing IdU, which has been attached using an automated batch stop-flow microwave apparatus. The utility of the Pd(0) cross-coupling to photosensitive pyrimidine is expanded herein to include the reaction of glass-supported ODN containing IdU under Heck and Suzuki conditions.
Subject(s)
Idoxuridine/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Palladium/chemistry , Pyrimidine Nucleosides/chemical synthesis , Pyrimidine Nucleosides/chemistryABSTRACT
We describe a novel reversible DNA interstrand photocrosslinking reaction via the artificial nucleoside (X). Oligodeoxynucleotide (ODN) containing X can be photocrosslinked by irradiation at 366 nm for 1 s, and the photocrosslinked ODN can be split by irradiation at 312 nm for 60 s.