Non-Cross-Linking Aggregation of DNA-Carrying Polymer Micelles Triggered by Duplex Formation.

Colloidal behaviors of particles functionalized with biomolecules are generally complicated. This study describes that colloidal behaviors of double-stranded (ds) DNA-carrying polymer micelles are well controlled by altering the molar ratio of single-stranded (ss) DNA moiety in the dsDNA shell. ssDNA-carrying micelles composed of a poly( N-isopropylacrylamide) (PNIPAAm) core surrounded by a dense shell of ssDNAs were prepared through self-assembly of PNIPAAm grafted with ssDNA by incubating its solution above the lower critical solution temperature. Spontaneous, non-cross-linking aggregation of the micelles was triggered by DNA duplex formation on the surface. Comparison of the critical coagulation concentration of NaCl among a series of the DNA-carrying micelles revealed the relationship between the helical structure of the surface-bound DNA and the colloidal stability of the micelles. The electrophoretic mobility analysis of the micelles indicated that the duplex formation reduced the structural flexibility of the surface-bound DNA, thereby decreasing the interparticle entropic repulsion. It is also suggested that the augmented rigidity of the surface-bound DNA increases the number of terminal base pairs facing the solvent, which could lead to multiple blunt-end stacking interaction among the micelles. Therefore, small DNA molecules could be considered unique surface-modifiers capable of controlling interactions between the surfaces of materials.

Noncontact Monitoring of Respiration by Dynamic Air-Pressure Sensor.

We have previously reported that a dynamic air-pressure sensor system allows respiratory status to be visually monitored for patients in minimally clothed condition. The dynamic air-pressure sensor measures vital information using changes in air pressure. To utilize this device in the field, we must clarify the influence of clothing conditions on measurement. The present study evaluated use of the dynamic air-pressure sensor system as a respiratory monitor that can reliably detect change in breathing patterns irrespective of clothing. Twelve healthy volunteers reclined on a dental chair positioned horizontally with the sensor pad for measuring air-pressure signals corresponding to respiration placed on the seat back of the dental chair in the central lumbar region. Respiratory measurements were taken under 2 conditions: (a) thinly clothed (subject lying directly on the sensor pad); and (b) thickly clothed (subject lying on the sensor pad covered with a pressure-reducing sheet). Air-pressure signals were recorded and time integration values for air pressure during each expiration were calculated. This information was compared with expiratory tidal volume measured simultaneously by a respirometer connected to the subject via face mask. The dynamic air-pressure sensor was able to receive the signal corresponding to respiration regardless of clothing conditions. A strong correlation was identified between expiratory tidal volume and time integration values for air pressure during each expiration for all subjects under both clothing conditions (0.840-0.988 for the thinly clothed condition and 0.867-0.992 for the thickly clothed condition). These results show that the dynamic air-pressure sensor is useful for monitoring respiratory physiology irrespective of clothing.

Thermodynamics-based rational design of DNA block copolymers for quantitative detection of single-nucleotide polymorphisms by affinity capillary electrophoresis.

Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-base-mutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe's ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 10(6) M(-1). The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation.

Dumbbell-shaped DNA analytes amplified by polymerase chain reaction for robust single-nucleotide polymorphism genotyping by affinity capillary electrophoresis.

A sample preparation method was developed for single-nucleotide polymorphism (SNP) genotyping based on hybridization between a single-stranded DNA (ssDNA) analyte and an allele-specific oligonucleotide (ASO) probe. When the SNP site is located in the stable secondary structure, the folding of this analyte imposes kinetic penalties on the hybridization with the ASO probe. To address this issue, the sequence of the ssDNA analyte was converted from the original one so that the analyte exhibited a clear dumbbell-shaped structure composed of two stem-loop moieties and an unfolded probe-binding site. The as-prepared analyte was structurally favorable for hybridization with the ASO probe, irrespective of the original sequence and secondary structure of the analyte. The sequence conversion was easily achieved by polymerase chain reaction using forward and reverse primers having an additional sequence at the 5'-terminus. These ssDNA analytes were subjected to affinity capillary electrophoresis using a diblock copolymer probe composed of an ASO segment and a poly(ethylene glycol) segment. The 70-base dumbbell-shaped analytes with a single-base difference were clearly separated within 12 min, although the original ones exhibited almost no separation due to the undesired folding of the probe-binding site. This sample preparation method should open up a wide range of applications for the ASO probes in genetic analysis.

Estimation of binding constants of peptide nucleic acid and secondary-structured DNA by affinity capillary electrophoresis.

An affinity capillary electrophoresis method was developed to determine a binding constant between a peptide nucleic acid (PNA) and a hairpin-structured DNA. A diblock copolymer composed of PNA and polyethylene glycol (PEG) was synthesized as a novel affinity probe. The base sequence of the probe's PNA segment was complementary to a hairpin-structured region of a 60-base single-stranded DNA (ssDNA). Upon applying a voltage, the DNA hairpin migrated slowly compared to a random sequence ssDNA in the presence of the PNA probe. This retardation was induced by strand invasion of the PNA into the DNA hairpin to form a hybridized complex, where the PEG segment received a large amount of hydrodynamic friction during electrophoresis. The binding constant between the PNA probe and the DNA hairpin was easily determined by mobility analysis. This simple method would be potentially beneficial in studying binding behaviors of various artificial nucleotides to natural DNA or RNA.

Quantitative SNP genotyping by affinity capillary electrophoresis using PEG-oligodeoxyribonucleotide block copolymers with electroosmotic flow.

Quantitative SNP detection was demonstrated with an ACE using a PEG-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as a probe in the presence of an EOF. The probe's PEG segment with large molecular weight and small polydispersity yielded a high resolution in the separation of a chemically synthesized 60-base ssDNA (WT) and its single-base-substituted mutant (MT). A mixture of WT and MT was clearly separated within 10 min by simultaneously using two types of PEG-b-ODN probes whose ODN segments were complementary to WT and MT and whose PEG segments were of different lengths. The peak area ratio between WT and MT was in good agreement with the feed ratio. The averaged difference between the feed and observed ratio of MT was determined to be 0.23%, which is lower than that of any other methods. The ACE using the PEG-b-ODN probes in the presence of EOF could be utilized as a facile method for estimating SNP allele frequency in various research fields.

Thermoresponsive micellization and micellar stability of poly(N-isopropylacrylamide)-b-DNA diblock and miktoarm star polymers.

Linear and miktoarm star-shaped diblock copolymers consisting of single-stranded DNA and poly(N-isopropylacrylamide) (PNIPAAm) with various compositions were synthesized via atom transfer radical polymerization and click chemistry. The temperature-responsive phase transition behavior, micellization, was systematically examined using UV-vis spectrometry, high-sensitivity differential scanning calorimetry, dynamic light scattering, and small-angle X-ray scattering. The lower critical solution temperature (LCST) increased, and its enthalpy decreased with decreasing PNIPAAm content. The copolymers self-assembled into well-defined nanoparticles having a core composed of PNIPAAm and a coronal layer of DNA above LCST. The particle size and micellar aggregation number of copolymer chains depended on the macromolecular composition and chain architecture. On the other hand, regardless of their factors, the surface area occupied by one DNA strand was found to be almost unchanged. The hybridization of DNA on the nanoparticles with fully complementary one induced the aggregation of the particles in a non-cross-linking configuration. The nanoparticle composed of miktoarm star copolymer showed a quicker DNA-hybridization response in this non-cross-linking aggregation compared with the case of a linear analogue.

RAFT-generated polyacrylamide-DNA block copolymers for single-nucleotide polymorphism genotyping by affinity capillary electrophoresis.

Capillary electrophoretic separation of a mixture of 5'-fluorescein isothiocyanate-labeled single-stranded DNA (normal ssDNA) and its single-base-substituted one (mutant ssDNA) was achieved by using a RAFT-generated polyacrylamide-oligodeoxyribonucleotide block copolymer (PAAm-b-ODN) as an affinity polymeric probe. PAAm-b-ODN was synthesized through the Michael addition of thiol-terminated PAAm (PAAm-SH) to 5'-maleimide-modified ODN. PAAm-SH was derived from dithiobenzoate-terminated PAAm prepared via RAFT polymerization. The number-averaged molecular weight (M(n)) and the molecular weight distribution were determined by aqueous size exclusion chromatography. After a capillary tube was filled with the running buffer solution of PAAm-b-ODN, a mixture of normal and mutant ssDNA was subjected to electrophoresis and detected by a laser-induced fluorescent detector. Because the base sequence of PAAm-b-ODN was complementary to part of the mutant ssDNA, including a single-base substitution site, the electrophoretic migration of mutant ssDNA was retarded due to the formation of the equilibrium complex with PAAm-b-ODN. Increasing M(n) of the PAAm segment enhanced this retardation. On the other hand, normal ssDNA was unable to form the complex owing to a single-base mismatch, which was proved by melting curve measurements. The Lineweaver-Burk-type analysis of the mobility of mutant ssDNA revealed that the binding constants for the complexes with different PAAm-b-ODN probes were almost identical to each other. The analysis also demonstrated that the ratio of the hydrodynamic radius of the complex to that of the free mutant ssDNA increased with increasing M(n) of the affinity polymeric probe's PAAm segment. This means that the PAAm segment indirectly provides mutant ssDNA with an additional hydrodynamic friction force via the affinity interaction of the ODN segment. Optimization of the salt concentration of the running buffer and the capillary temperature improved the resolution of the separation. This affinity polymeric probe will be useful for developing a simple and highly reliable single-nucleotide polymorphism genotyping method.

Affinity capillary electrophoretic DNA separation using PEG-oligodeoxyribonucleotide block copolymers: relationship between peak resolution and affinity strength.

Capillary electrophoretic separation of chemically synthesized ssDNA and a single-base-substituted one (normal and mutant ssDNA, respectively) was demonstrated using a PEG-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. When the base sequence of PEG-b-ODN was designed to be complementary to part of normal ssDNA including the base-substituted site, the electrophoretic mobility of normal ssDNA significantly decreased whereas that of mutant ssDNA slightly changed. Resolution of the separation strongly depended on the ODN length of the copolymer, the capillary temperature, and the Mg2+ concentration in the running buffer, indicating that the retardation of migration of normal ssDNA was induced by the reversible hybridization with PEG-b-ODN. It was found that the dissociation constant (K(d)) of the duplex between normal ssDNA and the affinity probe ODN should be smaller than 10(-6) M to achieve the good peak separation. In addition, we calculated the mobility of the complex (mu(C)) between normal ssDNA and PEG-b-ODN using a two-state model. The base sequence of affinity probe ODN appropriate to achieve the sufficient resolution will be predicted on the basis of the mu(C) and K(d )values.

Reversible Shrinkage of DNA-Functionalized Gold Nanoparticle Assemblies Revealed by Surface Plasmon Resonance.

A technique for tuning interparticle distance in plasmonic gold nanoparticle (AuNP) assemblies has shown great potential for the development of optoelectronic nanodevices. However, it still remains a challenge to reversibly alter the distance in a facile manner. DNA-templated AuNP assemblies are among the mostly investigated plasmonic nanomaterials. In previous work, salt-induced structural shrinkage of DNA-templated AuNP (5 nm in diameter) dimers/trimers is demonstrated only by electron microscopic analyses. In the present study, interparticle distance is modulated in larger AuNP (15 nm or 20 nm in diameter) dimers that exhibit strong surface plasmon resonance (SPR). The reversible SPR shift is achieved by using the temperature-dependent shrinkage/extension of the DNA-templated AuNP dimers. The present proof-of-concept study suggests a potential application of the reversible structural change to optical switching.

Turbidimetric detection of ATP using polymeric micelles and DNA aptamers.

Two types of turbidimetric detection of adenosine 5'-triphosphate (ATP) by the naked eye were achieved through a combination of non-cross-linking aggregation of DNA-linked polymeric micelles and molecular recognition of ATP by a DNA aptamer.

An alternative approach to the monitoring of respiration by dynamic air-pressure sensor.

Monitoring and assessing of patient respiratory function during conscious sedation are important because many drugs used for conscious sedation produce respiratory depression and subsequent hypoventilation. The purpose of this study is to assess the value of a dynamic air-pressure sensor for respiratory monitoring of clothed patients. Eight clothed adult volunteers were reclined on a dental chair positioned horizontally. The air bag for measuring air-pressure signals corresponding to respiration was placed on the seat back of the dental chair in the central lumbar area of the subject. The subject breathed through a face mask with a respirometer attached for measuring expiratory tidal volume. The air-pressure signals corresponding to respiration were obtained and the time integration values for air pressure during each expiration (integral P(exp)) were calculated. The expiratory tidal volume (TV(exp)) was measured simultaneously by respirometer. The relationship between TV(exp) and integral P(exp) for each subject was assessed by a Pearson correlation coefficient. A strong correlation between TV(exp) and integral P(exp) was observed in all subjects. Measuring integral P(exp) by dynamic air-pressure sensor makes it possible to estimate respiratory volume breath by breath, and the respiratory pressure-time integral waveform was useful in visually monitoring the respiration pattern. We believe that in the future this device will be used to monitor respiratory physiology in clothed patients, contributing to safer sedative procedures.

Capillary electrophoretic discrimination of single nucleotide polymorphisms using an oligodeoxyribonucleotidepolyacrylamide conjugate as a pseudo-immobilized affinity ligand: optimum ligand length predicted by the melting temperature values.

We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the Tm value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg2+ in the buffer solution.

Clinical recovery time from conscious sedation for dental outpatients.

For dental outpatients undergoing conscious sedation, recovery from sedation must be sufficient to allow safe discharge home, and many researchers have defined "recovery time" as the time until the patient was permitted to return home after the end of dental treatment. But it is frequently observed that patients remain in the clinic after receiving permission to go home. The present study investigated "clinical recovery time," which is defined as the time until discharge from the clinic after a dental procedure. We analyzed data from 61 outpatients who had received dental treatment under conscious sedation at the Hiroshima University Dental Hospital between January 1998 and December 2000 (nitrous oxide-oxygen inhalation sedation [n = 35], intravenous sedation with midazolam [n = 10], intravenous sedation with propofol [n = 16]). We found that the median clinical recovery time was 40 minutes after nitrous oxide-oxygen sedation, 80 minutes after midazolam sedation, and 52 minutes after propofol sedation. The clinical recovery time was about twice as long as the recovery time described in previous studies. In a comparison of the sedation methods, clinical recovery time differed (P = .0008), being longer in the midazolam sedation group than in the nitrous oxide-oxygen sedation group (P = .018). These results suggest the need for changes in treatment planning for dental outpatients undergoing conscious sedation.

Effect of a rotation training system on the mental health status of postgraduate dental trainees at Kyushu University Hospital, Fukuoka, Japan.

In Japan, the increasing frequency of mental health problems in postgraduate dental trainees has recently become apparent. To our knowledge, there has been no previous research to investigate the influence of the type of training program on the mental health of dental residents during one year of postgraduate clinical training. Therefore, the purpose of this study was to compare changes in the mental health of two groups of dental trainees at Kyushu University Hospital, Fukuoka, Japan: those who undertook a rotation training program and those who trained solely in one department (the control group). Study subjects in both groups completed the Profile of Mood States (POMS) and the General Health Questionnaire (GHQ) at five intervals throughout the postgraduate training year. Analysis of the questionnaire responses were performed by Student's t-test, analysis of variance, Bonferroni's test, and the chi-square test. Statistical tests showed differences in the mean scores of POMS-30 subscales and GHQ-28. The mood of anger was the factor that seemed to best describe the trainees' response to stress. The study results led to the conclusion that dental trainees' mental health is influenced by the type of training program and that dental trainees in rotation training programs may need more mental health support.

pH-responsive release of proteins from biocompatible and biodegradable reverse polymer micelles.

A reverse polymer micelle with a diameter of 100nm was prepared for a protein carrier releasing payloads in a pH-dependent manner. The reverse polymer micelle was made from an amphiphilic diblock copolymer of biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) and biocompatible poly(ethylene glycol) (PEG). PLGA having a terminal carboxyl group was additionally embedded in the micelle's PLGA layer via hydrophobic interaction. The micelles encapsulating bovine serum albumin and streptavidin released the proteins under neutral and basic conditions, whereas the proteins remained in the interior at acidic pH. Using erythropoietin as a protein drug, it was also exemplified that the released protein retained its cell proliferation activity even after rigorous formulation processes, including water-in-oil emulsion. The present reverse polymer micelle could potentially find application as an oral protein drug delivery carrier.

Preparation of cell-culturing glass surfaces that release branched polyethyleneimine triggered by thiol-disulfide exchange.

To develop a chemical stimulus-responsive substrate for culturing cells, polyethyleneimine (PEI) having a pyridyl disulfide moiety was attached via disulfide linkages to a glass coverslip modified with a silane coupling agent having a thiol group. The surface modification was confirmed by X-ray photoelectron spectroscopy and zeta potential analysis. The obtained surface exhibited sufficiently high cell adhesiveness. Zeta potential measurements revealed that the PEI derivatives were released from the surface through thiol-disulfide exchange when the modified glass coverslip was immersed in a neutral pH buffer containing cysteine. The cell viability assay demonstrated that this chemical stimulus was substantially nontoxic to 293T cells. Because PEI is a widely used transfection reagent, this functional glass coverslip would be potentially useful as an experimental platform for reverse transfection.

DNA terminal mismatch-induced stabilization of polymer micelles from RAFT-generated poly(N-isopropylacrylamide)-DNA block copolymers.

Temperature-responsive diblock copolymers made of poly(N-isopropylacrylamide) (PNIPAAm) generated by reversible addition-fragmentation chain transfer (RAFT) polymerization and a single-stranded DNA (ssDNA) self-assembled into polymer micelles. The micelles consisted of the PNIPAAm core surrounded by the ssDNA corona with a hydrodynamic diameter up to 300 nm in an aqueous medium above the lower critical solution temperature. In a medium of high ionic strength, the formation of the fully matched duplex with the complementary ssDNA on the surface of the polymer micelles induced rapid and spontaneous aggregation. By contrast, the micelles remained dispersed under the identical conditions when single-base-substituted ssDNA was added to form the corresponding terminal-mismatched duplex on the micellar surface. This highly sequence-selective process took place irrespective of the size of the PNIPAAm core.

Structural characterization of nanoparticles from thermoresponsive poly(N-isopropylacrylamide)-DNA conjugate.

Poly(N-isopropylacrylamide) (PNIPAAm) grafted with single-stranded (ss) DNA conjugate (PNIPAAm-g-DNA) self-assembles above its lower critical solution temperature to form colloidal particles. When the ssDNA within the particle hybridizes with its complementary DNA, the particles aggregate above a certain threshold of salt concentration with drastically increased turbidity in solution. Detailed structural information of the particle was obtained mainly by small-angle X-ray scattering. The influence of copolymer composition on the morphology of particle and non-crosslinking aggregation was examined. The particle consists of hydrophobic PNIPAAm core surrounded by hydrophilic DNA strands. The increase in DNA fraction brought about a significant decrease in core size, whereas the shell thickness little changed and corresponded to the length of DNA. A structural model with a sticky potential was applied to the analysis of particle aggregate. This analysis provided that the particles aggregate while the coronal layers interpenetrate each other. The interaction between the particles was quantified in terms of the sticky potential and showed a trend to be influenced by the particle size rather than the graft density of DNA strands on the particle.

Evaluation of single-base substitution rate in DNA by affinity capillary electrophoresis.

Capillary electrophoretic separation of 60 mer single-stranded DNA (ssDNA) and a single-base-substituted ssDNA was demonstrated using a size- and composition-controlled poly(ethylene glycol)-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. Under appropriate conditions, PEG-b-ODN and ssDNA with a complementary sequence formed a reversible complex via hybridization during the electrophoresis, while the copolymer did not interact with the single-base-substituted ssDNA. The copolymer's PEG length determined the electrophoretic mobility of the ssDNA; upon formation of the complex, the electrically neutral PEG added hydrodynamic friction to ssDNA. Simultaneously using two types of PEG-b-ODN copolymers whose PEG segments were of different lengths, we achieved the complete separation of the 60 mer ssDNA, its single-base-substituted ssDNA, and impurities. This method was sensitive enough to quantify a slight amount (approximately 1%) of the single-base-substituted ssDNA. The present results suggest that our approach is applicable to quantitative detection of minor genotypes.