Loop nucleotides impact the stability of intrastrand i-motif structures at neutral pH.

The stability of i-motif structures at neutral pH is of interest due to the potential of these structures to impact gene expression. A systematic investigation of loop sequence and length revealed that certain loop nucleobases stabilize i-motif quadruplexes.

Bulge-like asymmetric heterodye clustering in DNA duplex results in efficient quenching of background emission based on the maximized excitonic interaction.

Asymmetric dye clusters with a single fluorophore (Cy3) and multiple quenchers (4'-methylthioazobenzene-4-carboxylate, methyl red, and 4'-dimethylamino-2-nitroazobenzene-4-carboxylate) were prepared. The dye and one-to-five quenchers were tethered through D-threoninol to opposite strands of a DNA duplex. NMR analysis revealed that the clusters with a single fluorophore and two quenchers formed a sandwich-like structure (antiparallel H-aggregates). The melting temperatures of all the heteroclusters were almost the same, although structural distortion should become larger, as the number of quenchers increased. An asymmetric heterocluster of a single fluorophore and two quenchers showed larger excitonic interaction (i.e., hypochromicity of Cy3), than did a single Cy3 and a single quencher. Due to the larger exciton coupling between the dyes, the 1:2 heterocluster suppressed the background emission more efficiently than the 1:1 cluster. However, more quenchers did not enhance quenching efficiency due to the saturation of exciton coupling with two quenchers. Finally, this asymmetric 1:2 heterocluster was introduced into the stem region of a molecular beacon (MB; also known as an in-stem MB) targeting the fusion site in the L6 BCR-ABL fusion gene. With this MB design, the signal/background ratio was as high as 68 due to efficient suppression of background emission resulting from the maximized excitonic interaction.

Reversed assembly of dyes in an RNA duplex compared with those in DNA.

We prepared reversed dye clusters by hybridizing two RNA oligomers, each of which tethered dyes (Methyl Red, 4'-methylthioazobenzene, and thiazole orange) on D-threoninols (threoninol nucleotides) at the center of their strands. NMR spectroscopic analyses revealed that two dyes from each strand were axially stacked in an antiparallel manner to each other in the duplex, and were located adjacent to the 3'-side of a natural nucleobase. Interestingly, this positional relationship of the dyes was completely the opposite of that assembled in DNA that we reported previously: dyes in DNA were located adjacent to the 5'-side of a natural nucleobase. This observation was also consistent with the circular dichroism of dimerized dyes in which the Cotton effect of the dyes (i.e., the winding properties of two dyes) was inverted in RNA relative to that in DNA. Further spectroscopic analyses revealed that clustering of the dyes on RNA duplexes induced distinct hypsochromicity and narrowing of the band, thus demonstrating that the dyes were axially stacked (i.e., H-aggregates) even on an A-type helix. On the basis of these results, we also prepared heterodimers of a fluorophore (thiazole orange) and quencher (Methyl Red) in an RNA duplex. Fluorescence from thiazole orange was found to be strongly quenched by Methyl Red due to the excitonic interaction, so that the ratio of fluorescent intensities of the RNA-thiazole orange conjugate with and without its complementary strand carrying a quencher became as high as 27. We believe that these RNA-dye conjugates are potentially useful probes for real-time monitoring of RNA interference (RNAi) mechanisms.

A cationic dye triplet as a unique "glue" that can connect fully matched termini of DNA duplexes.

In this study, we propose that three consecutive cationic p-methylstilbazoles tethered on D-threoninols (Z residues) at 5' termini act as a unique "glue" connecting DNA duplexes by their interstrand cluster formation. Interstrand clustering of p-methylstilbazoles (ZZZ triplets) induces narrowing and hypsochromic shift of bands at 350 nm, which can be assigned to the absorption of p-methylstilbazole. However, single-stranded DNA conjugates involving a ZZZ triplet at the 5' terminus of 8-mer native nucleotides is found not to induce such large spectral changes, which implies that the intrinsic self-assembling property of ZZZ triplets is weak. Interestingly, when this conjugate is hybridized with a complementary 8-mer native oligonucleotide, a remarkable spectral change is observed, indicating the dimerization of a duplex through the interstrand clustering of ZZZ triplets. Dimerization of the duplex is also evidenced by cold-spray ionization mass spectrometry. This interstrand clustering is observed only when a ZZZ triplet is tethered to a 5' rather than 3' terminus. Furthermore, the stability of the interstrand cluster increases by increasing the number of nucleobases of the DNA portion, and when mismatched base pairs are incorporated or when a base next to the Z residue is deleted, the stability substantially drops. When we apply the ZZZ triplet to the formation of a nanowire using two complementary DNA conjugates, each of which has a ZZZ triplet at the 5' termini as overhang, we demonstrate the successful formation of a nanowire by native PAGE analysis. Since native sticky ends that have three nucleotides do not serve as "glue", ZZZ triplets with their unique glue-like properties are prime candidates for constructing DNA-based nanoarchitectures.

Gravity magnetic resonance imaging measurement of muscle pump change accompanied by aging and posture.

AIM: To date no age-comparative study has been reported about effect of exercise on muscle pump action change, while its effect is suggested to differ in ages. This study aims to clarify the changes in muscle pump action with aging by measuring the muscle and vein area, and blood flow in lower legs. METHODS: Subjects were healthy volunteers and consisted of three groups: young age group (N = 20), middle age group (N = 20) and old age group (N = 16). The lower leg flexor muscle area and popliteal vein area were measured by using T1-weighed magnetic resonance imaging at the condition pre- and post-ankle exercise in three positions. Moreover, popliteal blood flow velocity was also measured using phase contrast magnetic resonance imaging. RESULTS: The elderly had the highest number of individuals who had exercise habits (p < .001). In a multiple linear regression analysis, sitting posture, leg muscle volume, and rate of change in the soleus muscle were significantly related to blood flow velocity change. CONCLUSIONS: No difference was found in the changes in muscle pump action with age. The study results suggested that elderly people with exercise habits might be able to maintain the muscle pump action.

Anatomically safe sites for intramuscular injections: a cross-sectional study on young adults and cadavers with a focus on the thigh.

The anatomical safety of intramuscular injections at the deltoid and ventrogluteal sites has been investigated; however, the anatomical relationship between intramuscular injection sites in the thigh and major blood vessels and nerves remains unclear. We aimed to compare intramuscular injection sites in the rectus femoris and vastus lateralis with those at the deltoid and ventrogluteal sites and identify safe intramuscular injection sites in the thigh. Twenty-seven young adult volunteers were recruited, and the thicknesses of subcutaneous tissue and muscle as well as the number of blood vessels present were evaluated at two sites on the deltoid, ventrogluteal, and thigh using ultrasound equipment. The right thighs of 24 cadavers were used, and the thickness of muscle, number of blood vessels or nerves present, and the distance between each examined site and major blood vessels or nerves were evaluated in the rectus femoris and vastus lateralis. A major blood vessel was observed in the middle of the rectus femoris in young adults. In cadavers, the descending branch of the lateral circumflex femoral artery and muscle branch of the femoral nerves to the vastus lateralis were observed at the middle point, distal two-thirds point, and middle point between the middle and distal two-thirds points of the rectus femoris, but not at the middle of the vastus lateralis. The middle of the vastus lateralis is an appropriate site for intramuscular injections because of the low risk of vascular or nerve damage. The present results support good practices for site selection for intramuscular injections.

Factors affecting superficial vein visibility at the upper limb in healthy young adults: A cross-sectional observational study.

OBJECTIVE: Venipuncture is an invasive procedure, and repeated puncture attempts may be uncomfortable or even traumatic for patients. Vein visibility is one of the most influential variables for the failure of venipuncture; however, the factors affecting vein visibility remain unclear. The present study was conducted to identify the factors influencing vein visibility at the upper limb in healthy young adults. METHODS: Twenty-seven healthy volunteers were included. All measurements were performed at the right arm, right cubital fossa, and right forearm. The depth and cross-sectional area of superficial veins were measured by ultrasonography. Skin color was assessed by a spectrophotometer and quantified according to Commission International d'Eclairage L*a*b* values. RESULTS: Invisible superficial veins were significantly deeper and had a larger cross-sectional area than visible superficial veins. Skin color b* of invisible superficial veins was significantly higher than that of visible superficial veins. Vein depth, skin color b*, and gender markedly affected superficial vein visibility at the upper limb. The cutoff for vein depth was 2.3 mm (area under the curve = 0.91). CONCLUSION: The present results confirmed that vein depth, skin color b*, and gender strongly influenced vein visibility at the upper limb. The cutoff for vein depth was 2.3 mm.

Positively charged base surrogate for highly stable "base pairing" through electrostatic and stacking interactions.

"Base pairs" of cationic dyes (p-methylstilbazole) were incorporated into oligodeoxyribonucleotides (ODNs). This "base pair" greatly stabilized the duplex through electrostatic and stacking interactions. The melting temperature of modified ODN was higher than those of neutral dyes and native base pairs. Further stabilization of the duplex was observed when the number of cationic dyes increased.

Analysis of coherent heteroclustering of different dyes by use of threoninol nucleotides for comparison with the molecular exciton theory.

To test the molecular exciton theory for heterodimeric chromophores, various heterodimers and clusters, in which two different dyes were stacked alternately, were prepared by hybridizing two oligodeoxyribonucleotides (ODNs), each of which tethered a different dye on D-threoninol at the center of the strand. NMR analyses revealed that two different dyes from each strand were stacked antiparallel to each other in the duplex, and were located adjacent to the 5'-side of a natural nucleobase. The spectroscopic behavior of these heterodimers was systematically examined as a function of the difference in the wavelength of the dye absorption maxima (Delta lambda(max)). We found that the absorption spectrum of the heterodimer was significantly different from that of the simple sum of each monomeric dye in the single strand. When azobenzene and Methyl Red, which have lambda(max) at 336 and 480 nm, respectively, in the single strand (Delta lambda(max) = 144 nm), were assembled on ODNs, the band derived from azobenzene exhibited a small hyperchromism, whereas the band from Methyl Red showed hypochromism and both bands shifted to a longer wavelength (bathochromism). These hyper- and hypochromisms were further enhanced in a heterodimer derived from 4'-methylthioazobenzene and Methyl Red, which had a much smaller Delta lambda(max) (82 nm; lambda(max) = 398 and 480 nm in the single-strand, respectively). With a combination of 4'-dimethylamino-2-nitroazobenzene and Methyl Red, which had an even smaller Delta lambda(max) (33 nm), a single sharp absorption band that was apparently different from the sum of the single-stranded spectra was observed. These changes in the intensity of the absorption band could be explained by the molecular exciton theory, which has been mainly applied to the spectral behavior of H- and/or J-aggregates composed of homo dyes. However, the bathochromic band shifts observed at shorter wavelengths did not agree with the hypsochromism predicted by the theory. Thus, these data experimentally verify the molecular exciton theory of heterodimerization. This coherent coupling among the heterodimers could also partly explain the bathochromicity and hypochromicity that were observed when the dyes were intercalated into the duplex.

Threoninol as a scaffold of dyes (threoninol-nucleotide) and their stable interstrand clustering in duplexes.

Functional molecules such as dyes (Methyl Red, azobenzene, and Naphthyl Red) were tethered on D-threoninol as base surrogates (threoninol-nucleotide), which were consecutively incorporated at the center of natural oligodeoxyribonucleotides (ODNs). Hybridization of two ODNs involving threoninol-nucleotides allowed interstrand clustering of the dyes on D-threoninol and greatly stabilized the duplex. When two complementary ODNs, both of which had tethered Methyl Reds on consecutive D-threoninols, were hybridized, the melting temperature increased proportionally to the number of Methyl Reds, due to stacking interactions. Clustering of Methyl Reds induced both hypsochromicity and narrowing of the band, demonstrating that Methyl Reds were axially stacked relative to each other (H-aggregation). Since hybridization lowered the intensity of circular dichroism peaks at the pi-pi* transition region of Methyl Red (300-500 nm), clustered Methyl Reds were scarcely wound in the duplex. Alternate hetero dye clusters could also be prepared only by hybridization of two ODNs with different threoninol-nucleotides, such as Methyl Red-azobenzene and Methyl Red-Naphthyl Red combinations. A combination of Methyl Red and azobenzene induced bathochromic shift and broadening of the band at the Methyl Red region due to the disturbance of exciton interaction among Methyl Reds. But interestingly, the Methyl Red and Naphthyl Red combination induced merging of each absorption band to give a single sharp band, indicating that exciton interaction occurred among the different dyes. Thus, D-threoninol can be a versatile scaffold for introducing functional molecules into DNA for their ordered clustering.

Effects of metal ions and cosolutes on G-quadruplex topology.

Topologies of G-quadruplexes depend on oligonucleotide sequences and on environmental factors, and the diversity of G-quadruplex topologies complicates investigation of functions of these nucleic acid structures. To investigate how metal ions and cosolutes regulate topologies of G-quadruplexes, we stabilized the antiparallel conformation by insertion of 2'-deoxyxanthosine and 8-oxo-2'-deoxyguanosine into selected positions of an oligonucleotide. Thermodynamic analyses of the oligonucleotide revealed that Na+ stabilized the antiparallel G-quadruplex, whereas K+ destabilized this topology. This result suggests that metal ions selectively stabilize G-quadruplex topologies with cavities between G-quartet planes of certain sizes. In the presence of KCl in 20wt% poly(ethylene glycol) with average molecular weight of 200, the antiparallel basket-type G-quadruplex conformation was not stabilized compared with the dilute condition. In the presence of NaCl, the cosolute did stabilize the G-quadruplex with respect to the dilute condition. The presented data show that metal ions and cosolutes regulate topologies of G-quadruplexes through mechanisms that depend on sizes of metal ion cavities and hydration states.

Structure and Dynamics of Electron Injection and Charge Recombination in i-Motif DNA Conjugates.

The dynamics of electron injection have been investigated in intramolecular i-motif conjugates possessing stilbenediether (Sd) and perylenediimide (PDI) chromophores separated by either four or six hemiprotonated cytosine C-C base pairs assembled with synthetic loops. Circular dichroism spectra are consistent with the formation of i-motif structures in the absence or presence of Sd and PDI chromophores. The fluorescence of the Sd chromophore is essentially completely quenched by neighboring C-C base pairs, consistent with their function as an electron donor and electron acceptor, respectively. However, the fluorescence of the PDI chromophore is only partially quenched. The dynamics of electron injection from singlet Sd into the i-motif and subsequent charge recombination has been determined by femtosecond transient absorption (fsTA) spectroscopy and compared with the results for electron injection and charge recombination in Sd-linked hairpins possessing cytosine-guanine (C-G) or 5-fluorouracil-adenine (F-A) base pairs. While charge injection is ultrafast (<0.8 ps) for the i-motifs, charge transport across the i-motif C-C base pairs to the PDI electron trap is not observed. The absence of electron transport is related to the structure of the stacked C-C base pairs in the i-motif.

Mechanism and Dynamics of Electron Injection and Charge Recombination in DNA. Dependence on Neighboring Pyrimidines.

The mechanism and dynamics of photoinduced electron injection and charge recombination have been investigated for several series of DNA hairpins. The hairpins possess a stilbenediether linker, which serves as an electron donor and base pair stems that possess different pyrimidine bases adjacent to the linker. Hairpins with adjacent thymine-adenine (T-A) base pairs undergo fast electron injection and relatively slow charge recombination with rate constants that are not strongly dependent upon the following base pair. Hairpins with adjacent cytosine-guanine (C-G) base pairs undergo reversible electron injection and much faster charge recombination than those with adjacent T-A base pairs. Hairpins with 5-fluorouracil or other halogenated pyrimidines in their first and second base pair undergo fast electron injection and multiexponential charge recombination. The difference in kinetic behavior for the different series of hairpins and its implications for the formation of long-lived charge-separated states are discussed and compared to results reported previously for other electron-donor chromophores.

A polycation-chaperoned in-stem molecular beacon system.

In the presence of poly(L-lysine)-graft-dextran, an in-stem molecular beacon involving three perylene-anthraquinone pairs in the stem region had a signal/background ratio of as high as 570. Response speed was also remarkable; equilibrium was attained within 5 minutes after addition of substrate DNA at 20 °C.

Preparation of coherent hetero clusters with threoninol scaffold.

New hetero aggregates where different dyes stacked alternately were prepared by hybridizing two DNAs, each of which tethered a different dye in the centre of strand. Spectral changes due to exciton coupling between different kinds of dyes were observed. Especially, hetero aggregates of Methyl Red and 2- nitro-4'-dimethylaminoazobenzene showed substantial narrowing of the band, demonstrating coherent coupling occurred in these aggregates.

Incorporation of cationic dyes into DNA for distinct stabilization of duplex.

In this study, cationic dyes (methylstilbazole) were introduced into ODN. When two complementary ODNs, both of which tethered this dye, were hybridized, the melting temperature drastically increased. The duplex was further stabilized by introducing multiple dyes.

Construction of novel dye aggregates based on "comb-type" sequence.

Novel dye aggregates ("comb-type" aggregates) was prepared by hybridizing modified oligodeoxyribo-nucleotides (ODNs), in which dyes were introduced consecutively. From NMR structural analysis, dye molecules were intercalated between base pairs and stacked in anti-parallel manner. When ODNs containing three Methyl Red moieties were hybridized, strong exciton coupling was observed. In addition, thermal stability of duplex was substantially enhanced due to the intermolecular stacking.

Preparation of "comb-type" hetero aggregates by DNA-dye conjugation.

New alternating hetero aggregates ((")comb-type(") hetero aggregates) were successfully prepared by hybridizing two single-stranded DNAs, each of which tethered different homo-dye aggregates in the centre of the strand. When excitonically inert dyes (Methyl Red and azobenzene) were alternately assembled, broadening of the spectrum was observed. On the other hand, alternating aggregates of Methyl Red and Naphthyl Red showed substantial narrowing of the band. Although the peak maxima of these two dyes were different, strong exciton coupling occurred.