8-Pyrenylvinyl Adenine Controls Reversible Duplex Formation between Serinol Nucleic Acid and RNA by [2 + 2] Photocycloaddition.

Photocontrol of duplex formation between the totally artificial serinol nucleic acid (SNA) and target RNA was made possible using a photoresponsive nucleobase 8-pyrenylvinyl adenine (PVA). PVA residues in SNA can be induced to undergo intrastrand [2 + 2] photocycloaddition by 455 nm light. Effective cycloreversion of the PVA photodimer results from irradiation with 340 nm light. These reactions occurred in high yield, rapidly, selectively, and reversibly. When the PVA-SNA/RNA duplex was irradiated with 455 nm light, almost complete dissociation of the duplex was attained, and 340 nm light restored duplex formation by cycloreversion. This is the first example of use of photocycloaddition and cycloreversion to photoregulate canonical duplex formation and dissociation reversibly at constant temperature. Thus, SNA bearing PVA residues have potential for use in photocontrollable biological tools targeting endogenous RNAs in cells as well as photodriven SNA machines.

Temporal and Reversible Control of a DNAzyme by Orthogonal Photoswitching.

The reversible switching of catalytic systems capable of performing complex DNA  computing operations using the temporal control of two orthogonal photoswitches is described. Two distinct photoresponsive molecules have been separately incorporated into a split horseradish peroxidase-mimicking DNAzyme. We show that its catalytic function can be turned on and off reversibly upon irradiation with specific wavelengths of light. The system responds orthogonally  to a  selection of irradiation wavelengths    and   durations of irradiation. Furthermore, the DNAzyme exhibits reversible switching and retains this ability throughout multiple switching cycles. We apply our system as a light-controlled 4:2 multiplexer. Orthogonally photoswitchable DNAzyme-based catalysts as introduced here have potential use for controlling complex logical operations and for future applications in DNA nanodevices.

Quantification of Loading and Laser-Assisted Release of RNA from Single Gold Nanoparticles.

Novel RNA-based technologies provide an avenue of possibilities to control the regulation of gene expression in cells. To realize the full potential of small interfering RNA (siRNA)-based therapy, efficient delivery vehicles and novel strategies for triggering release from carrier vehicles have to be developed. Gold nanoparticles (AuNPs) with sizes of ∼50-150 nm have the ability to accumulate in tumor tissue and can be transported across the membrane by endocytosis. Therefore, a laser-controlled oligonucleotide release from such particles is of particular interest. Here, we quantify the loading of specifically attached microRNA oligonucleotides (miRNA) onto single gold nanoparticles with diameters of 80, 100, 150, and 200 nm. We show that AuNPs have a curvature-dependent density of miRNA loading: the higher the curvature, the higher the loading density. Moreover, we demonstrate how one sensing strand of an RNA duplex can be dehybridized and hence released from the AuNP by heating the AuNP by irradiation with a near-infrared (NIR) laser. Laser-induced release is also demonstrated inside living cells. Together, these findings show that plasmonic nanoparticles with high curvatures are ideal carriers of oligonucleotides into cells, and their cargo can be released in a controlled manner by a thermoplasmonic mechanism. Importantly, this remotely controlled release strategy can be applied to any cargo attached to a plasmonic nanocarrier, on either the single particle or ensemble level.

Aptamer photoregulation in vivo.

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.

Reversible Gel-Sol Transition of a Photo-Responsive DNA Gel.

Stimuli-responsive DNA gels that can undergo a sol-gel transition in response to photo-irradiation provide a way to engineer functional gel material with fully designed DNA base sequences. We propose an X-shaped DNA motif that turns into a gel by hybridization of self-complementary sticky ends. By embedding a photo-crosslinking artificial base in the sticky-end sequence, repetitive gel-sol transitions are achieved through UV irradiation at different wavelengths. The concentration of the DNA motif necessary for gelation is as low as 40 µm after modification of the geometrical properties of the motif. The physical properties, such as swelling degree and diffusion coefficient, were assessed experimentally.

Control of DNA hybridization by photoswitchable molecular glue.

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

Reversible regulation of metallo-base-pair interactions for DNA dehybridization by ultrasound.

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.

The effect of microwave irradiation on DNA hybridization.

The effect of microwave irradiation on DNA/DNA hybridization has been studied under controlled power and temperature conditions. It was discovered that microwave irradiation led to the melting of double-stranded deoxyoligonucleotides well below their thermal melting temperature and independent of the length of the deoxyoligonucleotides. These observations indicate a specific interaction of microwaves with DNA, and have important implications in the chemical or enzymatic processing of DNA under microwave heating.

RNA bandages for photoregulating in vitro protein synthesis.

'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.

Control of DNA hybridization with photocleavable adducts.

Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity.

Reusing nylon membranes for radioactive hybridizations.

We describe a procedure for recycling nylon hybridization membranes, enabling their repeated use for radioactive Southern hybridization analysis of different DNA samples. Following hybridization and probe removal, nylon membranes containing covalently linked DNAs were treated with 0.55% sodium hypochlorite. This destroyed the DNA, thereby preventing it from participating in further hybridization and enabling the membranes to be used subsequently for binding new DNA samples. With this procedure, we were able to reuse a single membrane as many as 13 times, with no detectable loss in signal. This method was shown to be effective for membranes supplied by three different manufacturers.

Use of Gen-Probe and Bactec for rapid isolation and identification of mycobacteria. Correlation of probe results with growth index.

Gen-Probe culture confirmation tests (Gen-Probe, San Diego, CA) for Mycobacterium tuberculosis complex and Mycobacterium avium complex were performed on 276 mycobacterial isolates. All 138 M. tuberculosis complex isolates and 79 of 80 M. avium complex isolates were identified correctly. No falsely positive test results were obtained; 58 nontuberculous mycobacteria other than M. avium complex were negative by Gen-Probe. In a second phase of testing, Gen-Probe tests were performed using concentrates from 101 patient Bactec 12B cultures. Positive results by Gen-Probe tests were correlated with the growth index (GI) reading on the day of processing as well as the accumulated GI readings. For those 51 with high (greater than or equal to 999) final GIs, 40/40 (100%) M. tuberculosis complex isolates and 9/11 M. avium complex isolates were positive by Gen-Probe, and six other mycobacteria were negative. Of the 25 with moderate final readings (400 less than or equal to GI less than 999), 12/17 M. tuberculosis complex isolates and 1/1 M. avium complex isolates were correctly identified by Gen-Probe; seven other mycobacteria were negative. Of 25 with low readings (GI less than 400), 8/24 M. tuberculosis isolates were correctly identified by Gen-Probe, and no falsely positive test results were obtained with the other probes. All true negative tests on seven other mycobacteria (not M. tuberculosis complex or M. avium complex) had less than 2% hybridization. Of the 24 falsely negative tests on M. tuberculosis complex isolates or M. avium complex isolates, 22 had greater than 2% hybridization with their respective probes. Thus, percent hybridization greater than 2% may be a useful indicator of the need for retesting.

Kinetics of recombinational hybrid formation in X-irradiated mammalian cells: a possible first step in the repair of DNA double-strand breaks.

It is known that mammalian cells repair X-ray-induced double-strand breaks (DSB). The mechanism of this repair is, however, as yet unknown but it is thought that the repair may involve recombination between homologous DNA strands. We have investigated the presence and kinetics of occurrence of recombinational molecular intermediates or 'heavy-heavy' (HH) hybrids in DNA of X-irradiated mouse Ehrlich ascites tumour cells unifiliarly substituted with bromodeoxyuridine. Purified DNA from density-labelled cells was analysed using isopyknic CsCl density centrifugation. After rebanding of the high-density material, the relative amount of HH-hybrid material was determined. When the cells were incubated after X-ray exposure, hybrids accumulated with an apparently biphasic kinetic; first a rapid accumulation directly after irradiation followed by a second, more slowly appearing peak. When DSB repair was inhibited by the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara-A), a powerful DNA polymerase inhibitor, the kinetics of the HH-hybrid formation were similar to those during the incubation after X-irradiation without ara-A. We interpret this as indicating that the first step in repair of DSB i.e. hybrid formation, occurs in the absence of DNA synthesis as predicted by the Resnick model of DSB repair. In an experiment in which the ara-A was washed away from the irradiated cells after 8-h treatment with the drug and replaced by fresh growth medium, so allowing DSB to repair, a similar HH-hybrid kinetic response was found to that occurring directly after irradiation in the absence of ara-A. The reasons for this are not yet clear. In this case, however, the response of the ara-A-treated cells after X-irradiation was much stronger than that in the untreated cells where only approximately 20% of the DSB remained. These kinetic results which show a temporary appearance of HH hybrids, indicate that a net exchange of genetic material does not take place; they therefore lend support to the postulated recombinational model of Resnick in which a temporary exchange between homologous DNA strands takes place during DSB repair.

Visible-light photocontrol of (E)/(Z) isomerization of the 4-(dimethylamino)azobenzene pseudo-nucleotide unit incorporated into an oligonucleotide and DNA hybridization in aqueous media.

We demonstrate significant photoresponsivity in aqueous media to visible light of pseudo-oligonucleotides possessing 4-(dimethylamino)azobenzene (4-DMAzo) side chains. The spectrum of the 4-DMAzo moiety during 436 nm light irradiation at pH >9 was clearly different from that of the all (E)-form, indicating the presence of the (Z)-form. Thermal (Z)-to-(E) recovery isomerization was faster at pH 9 (k(Z)(-E) = approximately 10(1) s(-1)) than at pH 11; however, addition of 50% ethanol significantly slowed the thermal recovery isomerization at pH 9 (k(Z)(-E) = approximately 2 s(-1)) and increased the magnitude of the spectral changes. Significant photoregulation of DNA hybridization by visible light was demonstrated under this condition.

Photoregulation of DNA hybridization by introducing an azobenzene: molecular design for more stabilization of DNA duplex with cis-azobenzene than with its trans-form.

Previously, photoregulation of DNA hybridization was achieved by introducing nonsubstituted azobenzene via a D-threoninol linker: DNA duplex formed (ON) after visible light irradiation (planar trans-form), whereas the duplex dissociated (OFF) after UV light irradiation (non-planar cis-form). In this study, for more efficient photoregulation of DNA functions, the reverse switch that can turn on duplex formation with UV, and turn off it with visible light irradiation was designed. When para-isopropylazobenzene (p-(i)PrAzo) was introduced into DNA via a L-thereoninol linker, the photoswitching direction was completely reversed: the duplex involving non-planar cis-p-(i)PrAzo was much more stable than that involving planar trans-form.

Incorporation of methyl group on azobenzene for the effective photo-regulation of hybridization and suppression of thermal isomerization.

We have synthesized azobenzene-tethered DNAs and have successfully photo-regulated various DNA functions. In the present study, we synthesized azobenzenes substituted with methyl group for still more effective photo-regulation of DNA hybridization. In trans-form, mono substituted azobenzene at ortho position stabilized the DNA duplex more efficiently than the other mono-substituted ones. In contrast, melting temperature (T(m)) for 2-methylazobenzene was lower in cis-form. As a result, change of T(m) (DeltaT(m)) induced by trans-cis isomerization became larger than that of unmodified azobenzene. Furthermore, di-substituted azobenzene at both ortho positions exhibited even larger DeltaT(m). Quite interestingly, thermal cis-to-trans isomerization of this azobenzene was about 10-fold slower than that of unmodified one. Thus, introduction of methyl group at 2, 6 positions raised both photo-regulatory activity and thermal stability of cis-form.

Porcine Parkin: molecular cloning of PARK2 cDNA, expression analysis, and identification of a splicing variant.

Parkin, encoded by the PARK2 gene, is an E3 ligase which functions as an integral component of the cytoplasmic ubiquitin/proteasomal protein degradation pathway. Mutations in the PARK2 gene, resulting in the loss of parkin function, leads to autosomal recessive juvenile Parkinsonism (AR-JP). This work reports the cloning and characterization of the porcine (Sus scrofa) PARK2 cDNA (SsPARK2) and splicing variants hereof. The PARK2 cDNA was amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine PARK2 cDNA codes for a protein of 461 amino acids which shows a high similarity to orangutan (91%), human (86%), and to rat (82%) parkin. A splicing variant of the porcine PARK2 with a complete deletion of exon 9 was also identified. Expression analysis by quantitative real-time RT-PCR revealed presence of PARK2 transcript in all examined organs and tissues. Differential expression was observed, with very high levels of PARK2 mRNA in cerebellum, heart, and kidney. In addition, expression analysis showed that porcine PARK2 transcripts could be detected early in embryo development in different brain regions. The porcine PARK2 orthologue was mapped to chromosome 1p24-25. Single nucleotide polymorphism (SNP) analysis revealed seven SNPs in the porcine PARK2 gene, one missense and one silent mutation in exon 7 and five SNPs in intron 7.

Interstrand cross-links: a new type of gamma-ray damage in bromodeoxyuridine-substituted DNA.

Interstrand cross-links (ICL) represent one of the most toxic types of DNA damage for dividing cells. They are induced both by natural products (e.g., psoralens + UVA) and by several chemical agents, some of which are used in chemotherapy (e.g., carboplatin and mitomycin C). Although repair mechanisms exist for interstrand cross-links, these lesions can induce mutations, chromosomal rearrangements, and cell death. Here, we report, for the first time, the formation of ICL by gamma-rays in brominated DNA. It is well established that the radiosensitization properties of bromodeoxyuridine (BrdUrd) result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of a uridinyl radical (dUrd(*)) or uridinyl anion (dUrd-) upon addition of an electron. We observe that the radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. We have used a model DNA containing a bulge, formed by five mismatched bases, and have observed a linear dose-response for the formation of strand breaks on the single-stranded regions of both the brominated strand and the opposite nonbrominated strand. Surprisingly, we have observed the formation of interstrand cross-links exclusively in the mismatched region. Thus, we propose that the radiosensitization effects of bromodeoxyuridine in vivo will almost certainly be limited to single strand regions such as found in transcription bubbles, replication forks, mismatched DNA, and possibly the loop region of telomeres. Our results suggest that interstrand cross-links may contribute to the radiosensitization effects of BrdUrd. These findings may have profound implications for the clinical use of bromodeoxyuridine as a radiosensitizer, as well as for the development of targeted radiosensitizers.

Alterations of liver DNA after x-irradiation. Reassociation and hybridization with nuclear RNA.

When RNA is extracted either from irradiated or unirradiated rat livers, no difference in its ability to hybridize with DNA is detectable. In contrast, the ability of DNA extracted from irradiated animals to hybirdize with RNA is decreased, probably as a result of DNA fragmentation. Sedimentation of X-irradiated DNA on sucrose gradients separates a small (low molecular weight) from a large peak (high molecular weight). The hybridization capacity for RNA of the large molecular weight DNA is similar to that of unirradiated DNA, but that of the small molecular weight is DNA reduced. After irradiation of the denatured DNA in vitro, the reassociation of DNA is inhibited, which suggests that DNA strands have lost part of their complementariness.