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We prepared microspheres densely covered with oligo-DNA strands by immobilizing amino-terminated oligo-DNA strands on the surface of carboxylate polystyrene latex (PS) particles via the amide bond formation. The obtained microspheres (ssDNA-PS) stably dispersed in neutral pH buffer containing high concentrations of NaCl. For the ssDNA-PS ≥1 μm diameter, only 3 - 5% of surface-immobilized oligo-DNA could form a duplex with the complementary strands. Nevertheless, the resulting ssDNA-PS showed a distinct duplex terminal dependency in their dispersion behavior under neutral pH and high NaCl conditions; the microspheres with fully-matched duplexes on the surface spontaneously aggregated in a non-crosslinking manner. By contrast, the microspheres with terminal-mismatched duplexes remained dispersed under the identical conditions. These results suggest that the micrometer-scale particles covered with oligo-DNA strands also have high susceptibility to a duplex terminal sequence in their dispersion property, similar to previously reported DNA-functionalized nanoparticles. This property could potentially be used in various applications including analytical purposes.

The interactions between metal ions and biomolecules are crucial to various bioprocesses. Development of plasmon switching nanodevices that exploit these molecular interactions is of fundamental and technological interest. Here, we show plasmon switching based on rapid aggregation/dispersion of double-stranded DNA-modified gold nanorods (dsDNA-AuNRs) that exhibit colloidal behaviors depending on pairing/unpairing of the terminal bases. The dsDNA-AuNRs bearing a thymine-thymine (T-T) mismatch at the penultimate position undergo spontaneous non-cross-linking aggregation in the presence of Hg 2+ due to T-Hg-T base pairing. Inversely, the subsequent addition of cysteine (Cys) gives rise to the removal of Hg 2+ from the T-Hg-T base pair to reproduce the T-T mismatch, resulting in stable dispersion of the dsDNA-AuNRs. The chemical-responsive plasmon switch allows for the rapid and repeatable cycles at room temperature. The validity of the present method is further exemplified by developing another plasmon switch fueled by Ag + and Cys by installing the Ag + -binding DNA sequence in the dsDNA-AuNR.

Hydrophobic attraction is often a physical origin of nonspecific and irreversible (uncontrollable) processes observed for colloidal and biological systems, such as aggregation, precipitation, and fouling with biomolecules. On the contrary, blunt-end stacking of complementary DNA duplex chain pairs, which is also mainly driven by hydrophobic interaction, is specific and stable enough to lead to self-assemblies of DNA nanostructures. To understand the reason behind these contradicting phenomena, we measured forces operating between two self-assembled monolayers of duplexed DNA molecules with blunt ends (DNA-SAMs) and analyzed their statistics. We found the high specificity and stability of blunt-end stacking that resulted in the high resemblance between the interaction forces measured on approaching and retracting. The other finding is on the stochastic formation process of blunt-end stacking, which appeared as a significant fluctuation of the interaction forces at separations smaller than 2.5 nm. Based on these results, we discuss the underlying mechanism of the specificity and stability of blunt-end stacking.

Directed assemblies of anisotropic metal nanoparticles exhibit attractive physical and chemical properties. However, an effective methodology to prepare differently directed assemblies from the same anisotropic nanoparticles is not yet available. Gold nanorods (AuNRs) region-selectively modified with different DNA strands can form side-by-side (SBS) and end-to-end (ETE) assemblies in a non-crosslinking manner. When the complementary DNA is hybridized to the surface-bound DNA, stacking interaction between the blunt ends takes place in the designated regions. Such AuNRs assemble into highly ordered structures, assisted by capillary forces emerging on the substrate surface. Moreover, insertion of a mercury(II)-mediated thymine-thymine base pair into the periphery of the DNA layer allows selective formation of the SBS or ETE assemblies from the strictly identical AuNRs with or without mercury(II)., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Double-stranded DNA-grafted nanoparticles (dsDNA-NPs) exhibit a unique dispersion behavior under high-salt conditions depending on the pairing status of their outermost base pairs (pairing or unpairing). The dsDNA-NPs having complementary (i.e., pairing) outermost base pairs spontaneously aggregate under high-salt conditions, but not when their outermost base pairs are mismatched (unpairing). In this study, we used colloidal probe atomic force microscopy to examine how the outermost base pairs affect the interaction between the dsDNA-grafted layers (dsDNA layers). To precisely assess the subtle structural differences in the dsDNA layers, we developed a method for the formation of a homogenous dsDNA layer on gold surfaces using hairpin-shaped DNAs. Homogenous dsDNA layers having complementary (G-C) or mismatched (C-C) outermost base pairs were grafted onto the surfaces of colloidal probes and gold substrates. Force-distance curves measured in an aqueous medium under high-salt conditions revealed that the surface forces between the dsDNA layers were bilateral in nature and were governed by the outermost base pairs. Between complementary outermost dsDNA layers, the surface force changed from repulsive to attractive with an increase in the NaCl concentration (10-1000 mM). The attraction observed under high-salt conditions was attributed to the site-specific interaction proceeded only between complementary dsDNA terminals, the so-called blunt-end stacking. In fact, between mismatched outermost dsDNA layers, the repulsive force was mostly dominant within the same NaCl concentration range. Our results clearly revealed that the pairing status of the outermost base pairs has significant implications for the surface forces between dsDNA layers, leading to the unique dispersion behavior of dsDNA-NPs.

Self-assembled structures of metallic nanoparticles with dynamically changeable interparticle distance hold promise for the regulation of collective physical properties. This paper describes gold nanoparticle dimers and trimers that exhibit spontaneous and reversible changes in interparticle distance. To exploit this property, a gold nanoparticle is modified with precisely one long DNA strand and approximately five short DNA strands. The long DNA serves to align the nanoparticles on a template DNA via hybridization, while the short DNAs function to induce the interparticle distance changes. The obtained dimer and trimer are characterized with gel electrophoresis, dynamic light scattering measurements, and transmission electron microscopy (TEM). When the complementary short DNA is added to form the fully matched duplexes on the particle surface in the presence of MgCl2 , spontaneous reduction of the interparticle distance is observed with TEM and cryo-electron microscopy. By contrast, when the terminal-mismatched DNA is added, no structural change occurs under the same conditions. Therefore, the single base pairing/unpairing at the outermost surface of the nanoparticle impacts the interparticle distance. This unique feature could be applied to the regulation of structures and properties of various DNA-functionalized nanoparticle assemblies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A single-nucleotide polymorphism (SNP) detection method was developed by combining single-base primer extension and salt-induced aggregation of gold nanoparticles densely functionalized with double-stranded DNA (dsDNA-AuNP). The dsDNA-AuNPs undergo rapid aggregation in a medium of high ionic strength, whereas particles having a single-base protrusion at the outermost surface disperse stably, allowing detection of a single-base difference in length by color changes. When SNP typing primers are used as analytes to hybridize to the single-stranded DNA on the AuNP surface, the resulting dsDNA-AuNP works as a visual indicator of single-base extension. A set of four extension reaction mixtures is prepared using each of ddNTPs and subsequently subjected to the aggregation assay. Three mixtures involving ddNTP that is not complementary to the SNP site in the target produce the aggregates that exhibit a purple color. In contrast, one mixture with the complementary ddNTP generates the single-base protrusion and appears red. This method could potentially be used in clinical diagnostics for personalized medicine., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

To estimate allele frequency of single-nucleotide polymorphisms (SNPs) in pooled DNAs with secondary structures, an affinity capillary electrophoresis was developed using an allele-specific peptide nucleic acid probe modified with polyethylene glycol. This probe disrupted secondary structures of DNA analytes and hybridized to them during electrophoresis. Such DNA-binding capability allowed separation of the folded analytes with a single-base difference within 20 min. The feed ratio of the target allele was evaluated by calculating the peak area ratio. The averaged difference between the feed and observed ratio was 1.5%. This method should be of general applicability to quantitative SNP analyses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Hybridization of DNA tethered on colloidal nanoparticles with fully matched complementary one induces the aggregation of the particles in a non-cross-linking configuration. Here, we performed a structural study on DNA-functionalized gold nanoparticle and its non-cross-linking aggregation mainly using synchrotron radiation small-angle X-ray scattering. To understand the non-cross-linking aggregation, the nanoparticles with various DNA lengths and core sizes were used. In the aggregation, the surface distance between the gold nanoparticles increased with the length of DNA duplex, although the increment of the distance per base pair was not constant and showed the tendency to become small with increasing DNA length, meaning the interdigitation of DNA layers. The aggregation was also found to occur between the identical cores, without being affected by tethered DNA. Furthermore, it was proved that the relative increase in DNA length to core size leads to the increase in colloidal stability. Even the nanoparticles with full-matched DNA duplex were dispersed stably. These facts suggested that van der Waals interaction between core particles rather than end-to-end stacking between DNA duplexes is a dominant attractive interaction. The steric repulsion force arising from entropic loss of thermal fluctuation of DNA molecules might be a key factor to characterize the non-cross-linking aggregation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Colorimetric detection of mercury ions (Hg(2+)) with the naked eye was accomplished within 1 min by a combination of non-crosslinking aggregation of double-stranded DNA-carrying gold nanoparticles and complex formation of thymine-Hg(2+)-thymine.

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.

A DNA-linked polymer micelle was prepared through a phase transition of an amphiphilic copolymer consisting of single-stranded DNA as the hydrophilic part and thermo-responsive poly(N-isopropylacryl-amide) as the hydrophobic part. Duplex formation on the surface of micelle decreased the colloidal stability, whereas a terminal single-base mismatch at the interface between the DNA shell and the disperse medium increased the colloidal stability. Based on this change, we successfully detected base pairing of 7,8-dihydro-8-oxo-2'-deoxyguanine with adenine. This method will be useful for discovering a Watson-Crick or non Watson-Crick base pair of an unknown oxidative-damaged base with each four canonical one.

DNA-modified gold nanoparticles exhibit a unique aggregation behaviour that they form aggregation with fully complementary DNA but do not with the terminal-mismatched DNA at certain concentration of added salts. We studied the aggregation structure of the nanoparticles by small angle X-ray scattering (SAXS). The results indicated that the gap between surfaces of gold nanoparticles is nearly two times as long as DNA in B-form. This suggests that the ends of completely double-stranded DNA (dsDNA) conjugated on gold nanoparticles contact or slightly interdigitate with each other in the aggregation structure.

alpha-Lactosyl-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) block copolymer (lactose-PEG-PAMA) was synthesized to construct a PIC micellar-type gene vector potentially useful for selective transfection of hepatic cells. Lactose-PEG-PAMA spontaneously formed a polyion complex (PIC) micelle with plasmid DNA (pDNA) encoding luciferase (pGL3-Luc) in aqueous solution without any precipitate formation. The lactosylated PIC micelle thus prepared achieved substantially higher transfection efficiency compared to the control PIC micelle without lactose moieties against HepG2 cells possessing asialoglycoprotein (ASGP) receptors recognizing the beta-d-galactose residue. This pronounced transfection efficacy of the lactosylated PIC micelle was inhibited by the addition of excess asialofetuin (ASF), a natural ligand against the ASGP receptor, indicating ASGP receptor-mediated endocytosis to be a major route of the cellular uptake of the lactosylated micelles. Notably, the lactosylated PIC micelle revealed enhanced transfection compared to the control PIC micelle at a lower dose of pDNA, demonstrating the feasibility of using the ligand-conjugated PIC micellar vector for gene delivery to targeted cells.