Optimizing the Kinetics and Thermodynamics of DNA i‐Motif Folding

Under slightly acidic conditions, single cytidine‐rich DNA strands can form four‐stranded structures called i‐motifs. The stability of the i‐motif structure is based on the intercalation of hemiprotonated C–C⁺ base pairs. In addition, the stability of these structures is influenced by pH, temperature, salt concentration, number of cytidines per C‐rich stretch, and length of sequence; it also depends on the nucleotides in the connecting loop regions. Here, we investigated the influence of the loop nucleotides on i‐motif stability, structure, and kinetics of folding, in five structures with the same loop‐size but different adenosine and thymidine residues within the loop. The stabilities of the i‐motif structures were determined by CD melting, and structure and kinetics of folding were studied by static and time‐resolved NMR experiments.     

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis,Hybridization Studies,Fluorescent Properties and i‐Motif Stability

The synthesis of two new phosphoramidite building blocks for the incorporation of 5‐(pyren‐1‐yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene‐modified nucleobase component were found to destabilize an i‐motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene‐modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene‐modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady‐state fluorescence emission studies of oligonucleotides containing pyrene‐modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene‐modified UNA monomers was observed after formation of i‐motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene‐modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.     

Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes

Enzyme-mediated proton transport across biological membranes is critical for many vital cellular processes. pH-sensitive fluorescent dyes are an indispensable tool for investigating the molecular mechanism of proton-translocating enzymes. Here, we present a novel strategy to entrap pH-sensitive probes in the lumen of liposomes that has several advantages over the use of soluble or lipid-coupled probes. In our approach, the pH sensor is linked to a DNA oligomer with a sequence complementary to a second oligomer modified with a lipophilic moiety that anchors the DNA conjugate to the inner and outer leaflets of the lipid bilayer. The use of DNA as a scaffold allows subsequent selective enzymatic removal of the probe in the outer bilayer leaflet. The method shows a high yield of insertion and is compatible with reconstitution of membrane proteins by different methods. The usefulness of the conjugate for time-resolved proton pumping measurements was demonstrated by using two large membrane protein complexes.     

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

We developed fluorescent turn‐on probes containing a fluorescent nucleoside, 5‐(benzofuran‐2‐yl)deoxyuridine (dU^BF) or 5‐(3‐methylbenzofuran‐2‐yl)deoxyuridine (dU^MBF), for the detection of single‐stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU^MBF achieved superior fluorescence enhancement than that containing dU^BF. NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3‐methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3‐methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex‐induced fluorescence enhancement of the oligonucleotide probe containing dU^MBF.     

Higher‐Order Human Telomeric G‐Quadruplex DNA Metalloenzymes Enhance Enantioselectivity in the Diels–Alder Reaction

Short human telomeric (HT) DNA sequences form single G‐quadruplex (G₄) units and exhibit structure‐based stereocontrol for a series of reactions. However, for more biologically relevant higher‐order HT G₄‐DNAs (beyond a single G₄ unit), the catalytic performances are unknown. Here, we found that higher‐order HT G₄‐DNA copper metalloenzymes (two or three G₄ units) afford remarkably higher enantioselectivity (>90 % ee) and a five‐ to sixfold rate increase, compared to a single G₄ unit, for the Diels–Alder reaction. Electron paramagnetic resonance (EPR) and enzymatic kinetic studies revealed that the distinct catalytic function between single and higher‐order G₄‐DNA copper metalloenzymes can be attributed to different Cu^II coordination environments and substrate specificity. Our finding suggests that, like protein enzymes and ribozymes, higher‐order structural organization is crucial for G₄‐DNA‐based catalysis.     

Chemical kinetic modeling of i‐butane and n‐butane catalytic cracking reactions over HZSM‐5 zeolite

A chemical kinetic model for i‐butane and n‐butane catalytic cracking over synthesized HZSM‐5 zeolite, with SiO₂/Al₂O₃ = 484, and in a plug flow reactor under various operating conditions, has been developed. To estimate the kinetic parameters of catalytic cracking reactions of i‐butane and n‐butane, a lump kinetic model consisting of six reaction steps and five lumped components is proposed. This kinetic model is based on mechanistic aspects of catalytic cracking of paraffins into olefins. Furthermore, our model takes into account the effects of both protolytic and bimolecular mechanisms. The Levenberg–Marquardt algorithm was used to estimate kinetic parameters. Results from statistical F‐tests indicate that the kinetic models and the proposed model predictions are in satisfactory agreement with the experimental data obtained for both paraffin reactants. © 2011 American Institute of Chemical Engineers AIChE J, 58: 2456–2465, 2012     

pH‐sensitive uniform gel beads for DNA adsorption

Uniform gel beads 3 mm in diameter were obtained by the suspension polymerization of an amine functionalized monomer, N‐3‐(dimethyl amino)propylmethacrylamide (DMAPM). The polymerization of DMAPM in the form of uniform droplets could be achieved at room temperature in an aqueous dispersion medium by using Ca–alginate gel as the polymerization mold. In this preparation, potassium persulfate/tetramethyl ethylenediamine and sodium alginate/calcium chloride were used as the redox initiator and the stabilizer systems, respectively. The crosslinked DMAPM gel beads exhibited pH‐sensitive, reversible swelling–deswelling behavior. The uniform gel beads were also obtained by the copolymerization of DMAPM and acrylamide (AA) in the same polymerization system. Although copolymer gel beads with higher pH sensitivities were obtained with increasing feed concentration of DMAPM, the total monomer conversion decreased. Crosslinked DMAPM and DMAPM–AA copolymer gel beads were utilized as sorbents for DNA adsorption. The gel beads produced with higher DMAPM feed concentration exhibited higher equilibrium DNA adsorption capacity. The DNA equilibrium adsorption capacities up to 50 mg DNA/g dry gel could be achieved with the crosslinked DMAPM gel beads. This value was reasonably higher relative to the previously reported adsorption capacities of known sorbents. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 3154–3161, 2000     

Functionalization of the 3′‐Ends of DNA and RNA Strands with N‐ethyl‐N‐coupled Nucleosides: A Promising Approach To Avoid 3′‐Exonuclease‐Catalyzed Hydrolysis of Therapeutic Oligonucleotides

The development of nucleic acid derivatives to generate novel medical treatments has become increasingly popular, but the high vulnerability of oligonucleotides to nucleases limits their practical use. We explored the possibility of increasing the stability against 3′‐exonucleases by replacing the two 3′‐terminal nucleotides by N‐ethyl‐N‐coupled nucleosides. Molecular dynamics simulations of 3′‐N‐ethyl‐N‐modified DNA:Klenow fragment complexes suggested that this kind of alteration has negative effects on the correct positioning of the adjacent scissile phosphodiester bond at the active site of the enzyme, and accordingly was expected to protect the oligonucleotide from degradation. We verified that these modifications conferred complete resistance to 3′‐exonucleases. Furthermore, cellular RNAi experiments with 3′‐N‐ethyl‐N‐modified siRNAs showed that these modifications were compatible with the RNAi machinery. Overall, our experimental and theoretical studies strongly suggest that these modified oligonucleotides could be valuable for therapeutic applications.     

Regulation of Poly-E Motif Flexibility by pH,Ca2+ and the PPAK Motif

The disordered PEVK region of titin contains two main structural motifs: PPAK and poly-E. The distribution of these motifs in the PEVK region contributes to the elastic properties of this region, but the specific mechanism of how these motifs work together remains unclear. Previous work from our lab has demonstrated that 28-amino acid peptides of the poly-E motif are sensitive to shifts in pH, becoming more flexible as the pH decreases. We extend this work to longer poly-E constructs, including constructs containing PPAK motifs. Our results demonstrate that longer poly-E motifs have a much larger range of pH sensitivity and that the inclusion of the PPAK motif reduces this sensitivity. We also demonstrate that binding calcium can increase the conformational flexibility of the poly-E motif, though the PPAK motif can block this calcium-dependent change. The data presented here suggest a model where PPAK and calcium can alter the stiffness of the poly-E motif by modulating the degree of charge repulsion in the glutamate clusters.     

Fluorescent DNA Labeling by N‐Mustard Analogues of S‐Adenosyl‐L‐Methionine

Azide and alkyne‐functionalized N‐mustard analogues of S‐adenosyl‐L ‐methionine have been synthesized and were demonstrated to undergo efficient methyltransferase‐dependent DNA alkylation by M.TaqI and M.HhaI. Subsequent labeling of the DNA with a fluorophore was carried out using copper‐catalyzed azide–alkyne cycloaddition chemistry and was visualized by fluorescence scanning. This work demonstrates the utility of functionalized N‐mustard analogues as biochemical tools to study biological methylation and offers a facile way to site‐selectively label substrates of DNA methyltransferases.     

Investigation of pH sensitivity of poly(acrylic acid‐co‐acrylamide) hydrogel

It was found that the upper critical solution temperature (UCST) of poly(acrylic acid‐co‐acrylamide) hydrogel decreases with increase in pH. The UCST of samples equilibrated in a buffer at pH 2.5 was 33.7 °C, whereas it shifted to below 0 °C when swollen in buffers above pH 10. FT‐IR showed that COOH group of acrylic acid ionized gradually in alkaline media. The morphology change of the hydrogel swollen in different buffers was studied by environmental scanning electron microscopy (ESEM). ESEM images showed that the pore size of the sample increased with increase in pH, and in strongly basic media the three‐dimensional network was replaced by a ‘sausage‐like’ or ‘desert‐like’ structure. A study of the pH‐dependent release of cefazolin sodium was also carried out. Copyright © 2003 Society of Chemical Industry     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Thermal and pH dual‐responsive hydrogels based on semi‐interpenetrating network of poly(N‐isopropylacrylamide) and collagen nanofibrils

Hydrogels with environment‐sensitive properties have great potential applications in the controlled drug release field. In this paper, hybrid hydrogels with semi‐interpenetrating polymer networks (semi‐IPNs), composed of poly(N‐isopropylacrylamide) (PNIPAM) as the thermo‐sensitive component by in situ polymerization and self‐assembled collagen nanofibrils as the pH‐sensitive framework, were prepared for controlled release of methyl violet as a model drug. From Fourier transform infrared spectroscopy and scanning electron microscopy, it was indicated that the crosslinking of PNIPAM in the presence of collagen nanofibrils led to the formation of semi‐IPNs with homogeneous porous structure, and the semi‐IPNs showed improved thermal stability and elastic properties compared with the native collagen as determined using differential scanning calorimetry and rheologic measurements. Furthermore, the semi‐IPNs possessed swelling behaviors quite different from those of neat collagen or PNIPAM hydrogel under various pH values and temperatures. Correspondingly, as expected, the drug release behavior in vitro for semi‐IPNs performed variously compared with that for single‐component semi‐IPNs, which revealed the tunable performance of semi‐IPNs for release ability. Finally the thermo‐ and pH‐responsive mechanism of the semi‐IPNs was illuminated to provide guidance for the application of the thermo‐ and pH‐sensitive collagen‐based hybrid hydrogels in controlled drug delivery systems. © 2019 Society of Chemical Industry     

Dopants dependent ion sensitivity of polypyrrole‐modified electrode: Case of pH sensing

The polypyrrole (PPY)‐modified electrode made through electropolymerization of pyrrole monomer is found sensitive to different cations depending on the dopants present within microstructure of polymeric domain. When p‐toluenesulfonate (PTS) anion is doped, the modified electrode is sensitive to proton whereas, under similar conditions, in the presence of tetraphenylborate (TPB) anion, the same is sensitive to zinc ion. A comparative study on ion sensing behavior of PPY‐modified electrodes is reported in this communication. The electro‐polymerization of PTS‐doped PPY‐modified electrode, made under both potentiodynamic and potentiostatic conditions, are studied. The modified electrode is characterized by scanning electron microscopy and cyclic voltammetry and compared with those recorded for TPB‐doped PPY‐modified electrode made under similar conditions. The results based on scanning electron microscopy and cyclic voltammetry suggested; (i) dependence of microstructure based on mode of electropolymerization; (ii) dopant‐dependant ion sensitivity of PPY‐modified electrode, (iii) variation in the redox behavior of the PPY‐modified electrode based on dopants and mode of electropolymerization. Typical results on pH sensing together stability of PPY‐based pH sensor are reported and compared with those recorded for polyaniline (PANI)‐based pH sensor. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Evaluation of UDP‐GlcN Derivatives for Selective Labeling of 5‐(Hydroxymethyl)cytosine

5‐(hydroxymethyl)cytosine (5‐hmC) is a newly identified oxidative product of 5‐methylcytosine (5‐mC) in the mammalian genome, and is believed to be an important epigenetic marker influencing a variety of biological processes. In addition to its relatively low abundance, the fluctuation of 5‐hmC levels over time during cell development poses a formidable challenge for its accurate mapping and quantification. Here we describe a specific chemoenzymatic approach to 5‐hmC detection in DNA samples by using new uridine 5′‐diphosphoglucosamine (UDP‐GlcN) probes. Our approach requires modification of the glucose moiety of UDP‐Glc with small amino groups and transfer of these glucose derivatives to the hydroxy moiety of 5‐hmC by using T4 phage glucosyltransferases. We evaluated the transfer efficiencies of three glucosyltransferases (wild‐type α‐ and β‐GTs and a Y261L mutant β‐GT) with five different UDP‐Glc derivatives containing functionalized groups for subsequent bioconjugation and detection. Our results indicate that UDP‐6‐N₃‐Glc, UDP‐6‐GlcN, and UDP‐2‐GlcN can be transferred by β‐GT with efficiencies similar to that seen with the native UDP‐Glc cofactor. 6‐N₃‐Glc‐ and 6‐GlcN‐containing oligonucleotides were selectively labeled with reactive fluorescent probes. In addition, a 2 kb DNA fragment modified with 2‐GlcN groups was specifically detected by use of a commercially available antiglucosamine antibody. Alternative substrates for β‐GT and correlated glycosyltransferases might prove useful for the study of the function and dynamics of 5‐hmC and other modified nucleotides, as well as for multiplex analysis.     

An optimization‐based approach for structural design of self‐assembled DNA tiles

DNA tiles are self‐assembled nanostructures, which offer exciting opportunities for synthesis of novel materials. A challenge for structural design of DNA tiles is to identify optimal locations for so‐called crossovers, which are bridges between DNA double helices formed by pairs of single‐stranded DNA. An optimization‐based approach is presented to identify optimal locations for such crossovers. Minimization of a potential‐energy model for a given structural design demonstrates the importance of local minima. Both deterministic global optimization of a reduced model and multistart optimization of the full model are applied successfully to identify the global minimum. MINLP optimization using a branch‐and‐bound algorithm (GAMS/SBB) identifies an optimal structural design of a DNA tile successfully with significant reduction in computational load compared to exhaustive enumeration, which demonstrates the potential of the proposed method to reduce trial‐and‐error efforts for structural design of DNA tiles. © 2016 American Institute of Chemical Engineers AIChE J, 63: 1804–1817, 2017