This document discusses tuning the pH response of i-motif DNA oligonucleotides. The authors introduced 5-methylcytosines (5-MeC) and 5-bromocytosines (5-BrC) into the human telomeric i-motif sequence to shift its pH response range. They found that 5-MeC shifted the pH response towards more basic values, while 5-BrC shifted it towards more acidic values. Additionally, lengthening the sequence shifted the pH response in a more basic direction. The modifications did not thermally destabilize the i-motifs. 5-BrC substitution led to a ten-fold increase in folding kinetics compared to the other sequences.
34th Annual Mid-West Photosynthesis Conference at Turkey Run State Park, INSean Padden
The document reports on a study of mutations to the arginine residue at position 257 (R257) of the D1 protein in Photosystem II in Chlamydomonas reinhardtii. Three mutations were made - R257E, R257K, and R257Q. Thermoluminescence and fluorescence measurements showed that the redox potential of the QB/QB- pair was lowered by 20-40 mV in the mutants. This suggests the size and charge of the residue at position 257 modulates the redox potential of QB/QB-. Growth rates and oxygen evolution were negatively impacted in the mutants compared to wild type.
The crystal structure of the N-terminal domain (RbN) of the retinoblastoma tumor suppressor protein was determined. RbN adopts a globular structure formed by two rigidly connected cyclin-like folds (lobes A and B). Structural analysis revealed similarities between RbN and the A and B boxes of the Rb pocket domain, suggesting Rb evolved through domain duplication. The structure provides insights into the oncogenicity of RbN mutations and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, the structure suggests a model of the full-length Rb protein where the RbN and pocket domains directly interact, and this interaction can be modulated by ligand binding
This study used NMR spectroscopy to measure hydrogen exchange rates of backbone amide protons in two disordered proteins, a-synuclein and FlgM, in buffer and inside Escherichia coli cells. The rates were similar in buffer and cells, and close to rates predicted from studies of unstructured peptides. This suggests that true disorder can persist inside the crowded cellular interior, and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic exchange rates. However, the C-terminal region of FlgM showed some evidence of transient structure formation in buffer that was not observed inside cells.
The document summarizes the synthesis and evaluation of ether lipid analogs of known TLR4 agonist CRX-527 and antagonist CRX-526. A new efficient synthesis in 11 steps utilizes a common advanced intermediate approach and yields the target compounds and analogs in high purity. In vitro and in vivo assays show that the ether analog of CRX-527 maintains potent agonist activity similar to CRX-527, while the ether analog of CRX-526 does not maintain antagonist activity and instead displays weak agonist properties in mouse models.
This document describes the crystal structure and characterization of a covalently cross-linked complex between cytochrome c peroxidase (CCP) and cytochrome c (cyt c) that was engineered by introducing cysteine mutations into the proteins. The 1.88 angstrom crystal structure of the cross-linked complex closely resembles the structure of the noncovalent complex and reveals ordered water molecules bridging the interface. Studies show the cross-linked complex maintains normal compound I formation and fast intramolecular electron transfer, indicating it closely mimics the physiological electron transfer complex.
This document describes research that used comparative sequence analysis to identify regions of the ribosome that have been evolutionarily conserved. The researchers determined the spatial organization of conserved domains by mapping them onto existing ribosomal structures. They found that several functional domains are conserved in their three-dimensional organization across Archaea, Bacteria, and Eukaryotes. However, some regions like the L11 binding domain and alpha-sarcin-ricin loop have shifted positions during evolution. The researchers also examined conserved interactions between the ribosomal subunits and tRNA contacts to identify functionally important interactions during protein synthesis. They conclude by summarizing findings from analyzing the small rRNA sequence of the C. elegans mitochondrion to provide insight into the evolution
This document summarizes research characterizing a chlorite dismutase (Cld) enzyme from Klebsiella pneumoniae. The enzyme, KpCld, is part of a subfamily of dimeric Clds found in non-perchlorate respiring bacteria. While it shares structural similarities in its active site with efficient O2-producing Clds, it exhibits limited turnover due to degradation of its heme cofactor. Experiments show KpCld can generate O2 from chlorite, and a K. pneumoniae mutant lacking Cld has reduced growth in the presence of chlorate under nitrate-respiring conditions, suggesting KpCld functions to detoxify endogenously produced chlorite. The
The document summarizes research on developing glycopeptide analogues of PSGL-1 that inhibit P-selectin. Key findings include:
1) Researchers developed an efficient multi-step synthesis of a C2 O-glycan building block, overcoming prior limitations.
2) Using the building block, they synthesized glycopeptide mimics of PSGL-1's N-terminus via solid phase peptide synthesis, replacing acid-labile tyrosine sulfates with stable sulfonates.
3) Screening identified a compound, GSnP-6, with nanomolar affinity for P-selectin that potently blocks P-selectin/PSGL-1 interactions in vitro and
34th Annual Mid-West Photosynthesis Conference at Turkey Run State Park, INSean Padden
The document reports on a study of mutations to the arginine residue at position 257 (R257) of the D1 protein in Photosystem II in Chlamydomonas reinhardtii. Three mutations were made - R257E, R257K, and R257Q. Thermoluminescence and fluorescence measurements showed that the redox potential of the QB/QB- pair was lowered by 20-40 mV in the mutants. This suggests the size and charge of the residue at position 257 modulates the redox potential of QB/QB-. Growth rates and oxygen evolution were negatively impacted in the mutants compared to wild type.
The crystal structure of the N-terminal domain (RbN) of the retinoblastoma tumor suppressor protein was determined. RbN adopts a globular structure formed by two rigidly connected cyclin-like folds (lobes A and B). Structural analysis revealed similarities between RbN and the A and B boxes of the Rb pocket domain, suggesting Rb evolved through domain duplication. The structure provides insights into the oncogenicity of RbN mutations and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, the structure suggests a model of the full-length Rb protein where the RbN and pocket domains directly interact, and this interaction can be modulated by ligand binding
This study used NMR spectroscopy to measure hydrogen exchange rates of backbone amide protons in two disordered proteins, a-synuclein and FlgM, in buffer and inside Escherichia coli cells. The rates were similar in buffer and cells, and close to rates predicted from studies of unstructured peptides. This suggests that true disorder can persist inside the crowded cellular interior, and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic exchange rates. However, the C-terminal region of FlgM showed some evidence of transient structure formation in buffer that was not observed inside cells.
The document summarizes the synthesis and evaluation of ether lipid analogs of known TLR4 agonist CRX-527 and antagonist CRX-526. A new efficient synthesis in 11 steps utilizes a common advanced intermediate approach and yields the target compounds and analogs in high purity. In vitro and in vivo assays show that the ether analog of CRX-527 maintains potent agonist activity similar to CRX-527, while the ether analog of CRX-526 does not maintain antagonist activity and instead displays weak agonist properties in mouse models.
This document describes the crystal structure and characterization of a covalently cross-linked complex between cytochrome c peroxidase (CCP) and cytochrome c (cyt c) that was engineered by introducing cysteine mutations into the proteins. The 1.88 angstrom crystal structure of the cross-linked complex closely resembles the structure of the noncovalent complex and reveals ordered water molecules bridging the interface. Studies show the cross-linked complex maintains normal compound I formation and fast intramolecular electron transfer, indicating it closely mimics the physiological electron transfer complex.
This document describes research that used comparative sequence analysis to identify regions of the ribosome that have been evolutionarily conserved. The researchers determined the spatial organization of conserved domains by mapping them onto existing ribosomal structures. They found that several functional domains are conserved in their three-dimensional organization across Archaea, Bacteria, and Eukaryotes. However, some regions like the L11 binding domain and alpha-sarcin-ricin loop have shifted positions during evolution. The researchers also examined conserved interactions between the ribosomal subunits and tRNA contacts to identify functionally important interactions during protein synthesis. They conclude by summarizing findings from analyzing the small rRNA sequence of the C. elegans mitochondrion to provide insight into the evolution
This document summarizes research characterizing a chlorite dismutase (Cld) enzyme from Klebsiella pneumoniae. The enzyme, KpCld, is part of a subfamily of dimeric Clds found in non-perchlorate respiring bacteria. While it shares structural similarities in its active site with efficient O2-producing Clds, it exhibits limited turnover due to degradation of its heme cofactor. Experiments show KpCld can generate O2 from chlorite, and a K. pneumoniae mutant lacking Cld has reduced growth in the presence of chlorate under nitrate-respiring conditions, suggesting KpCld functions to detoxify endogenously produced chlorite. The
The document summarizes research on developing glycopeptide analogues of PSGL-1 that inhibit P-selectin. Key findings include:
1) Researchers developed an efficient multi-step synthesis of a C2 O-glycan building block, overcoming prior limitations.
2) Using the building block, they synthesized glycopeptide mimics of PSGL-1's N-terminus via solid phase peptide synthesis, replacing acid-labile tyrosine sulfates with stable sulfonates.
3) Screening identified a compound, GSnP-6, with nanomolar affinity for P-selectin that potently blocks P-selectin/PSGL-1 interactions in vitro and
This document summarizes research on converting an engineered potassium-binding site in cytochrome c peroxidase (CCP) into a calcium-selective site through protein engineering and crystal structure analysis. The researchers previously engineered a potassium-binding site in CCP based on the structure of ascorbate peroxidase. They then designed mutants intended to bind calcium selectively instead. The crystal structure of the first mutant showed binding of a smaller cation like sodium rather than calcium due to disordering of a ligand. A second mutant was then designed and its crystal structure confirmed calcium binding with a fully coordinated ligand environment, demonstrating that an iterative engineering approach can switch cation selectivity in proteins.
Biomimetic Glycoside Hydrolysis by a Microgel Templated with a Competitive Gl...aaaa zzzz
This document summarizes the synthesis and characterization of biomimetic microgel catalysts templated with a competitive glycosidase inhibitor (galactonoamidine). The microgels were synthesized via free radical polymerization in the presence of a binuclear copper complex and the inhibitor. Computational modeling was used to determine the inhibitor binds to the copper complex through its amidine and hydroxyl groups. The resulting microgels were characterized and found to have a narrow size distribution and near-quantitative incorporation of the copper complex. The microgels show potential as biomimetic catalysts for glycoside hydrolysis.
Insertions and deletions are undervalued and underutilized source of diversity. Below is an example how a deletion can change metal selectivity of the CbiX chelatase.
The document summarizes characterization of the H134C mutant of the Thermus thermophilus Rieske protein. Key findings include:
1) The H134C mutant substitutes one ligating histidine for a cysteine, changing the iron-sulfur cluster ligation from 2Cys-2His to 3Cys-1His.
2) Studies using UV-visible spectroscopy, circular dichroism, and reaction with diethyl pyrocarbonate (DEPC) show the H134C mutant is stable across a wide pH range, unlike the similar mitochondrial protein mitoNEET.
3) Reaction of H134C with DEPC results in modification of the cluster but no reduction,
Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regul...Wei Fang
This document summarizes a study that used RNA sequencing to analyze changes in gene expression in the alga Chlamydomonas reinhardtii under different carbon dioxide (CO2) conditions and in a mutant lacking the CO2-concentrating mechanism regulator CIA5. The study found massive effects on the transcriptome, with almost 25% of genes affected. Distinct clusters of genes were identified that responded primarily to either CIA5 or CO2. Several clusters associated with CO2-concentrating mechanism genes were identified that may contain new candidate genes involved in inorganic carbon transport.
This document summarizes research exploring the biochemical properties and remediation applications of an unusual P450 system (XplA/B) found in Rhodococcus bacteria that is able to degrade the explosive compound RDX. Key findings include:
1) XplA has a high affinity for RDX (Kd = 58 μM) and XplB functions as its native reductase partner to efficiently degrade RDX.
2) Expression of both XplA and XplB in Arabidopsis plants enables significantly faster removal of RDX from contaminated liquid culture and soil compared to plants expressing only XplA.
3) Under anaerobic conditions, RDX degradation by XplA/B produces
1) Cryo-EM was used to determine the structure of the human nucleosome containing the histone variant H2A.B (H2A.B-NCP) at atomic resolution of 3.9 Angstroms.
2) The H2A.B-NCP structure is distinct from canonical nucleosomes, compacting around 103 base pairs of DNA in 1.2 superhelical turns compared to 145 base pairs in 1.7 turns for canonical nucleosomes.
3) A poly(ADP-ribose) polymerase 1 DNA binding domain was found to stabilize the dynamic H2A.B-NCP, allowing determination of its structure at higher resolution of 2.8 Ang
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
This document presents a structure/function model for the enzyme 1-pyrroline-5’ carboxylate reductase (P5CR) based on similarities to the β-hydroxyacid dehydrogenase enzyme family. The model is supported by evidence that recombinant E. coli P5CR has similar secondary structure to a β-hydroxyacid dehydrogenase and contains conserved functional domains. Site-directed mutagenesis of conserved residues in the proposed substrate-binding domain reduced catalytic efficiency. Chemical modification studies also provided insights into active site residues. The model proposes γ-glutamate semialdehyde as a potential true substrate based on inhibition studies.
2014. Pietkiewicz, Wahyudi. Synthesis of macrocycles that inhibit protein syn...Adrian Pietkiewicz
This document describes the synthesis of seven new analogues of the macrocyclic peptide sanguinamide B (SanB) and testing of their ability to inhibit protein synthesis in cancer cells. The analogues were designed by altering the amino acids at positions I and III of the SanB backbone, inverting stereochemistry at position III, and changing the protecting group on lysine. All analogues were tested for cytotoxicity against colon cancer cell lines and ability to inhibit protein synthesis. The lead compound with an IC50 of 15.9 μM against colon cancer cells contained an N6-carboxybenzyl-lysine at position I. This establishes the importance of this moiety for biological activity.
This document appears to be a midterm exam for a university course on biochemistry. It contains multiple choice and true/false questions about topics like glycolysis, gluconeogenesis, the citric acid cycle, and related metabolic pathways. It also asks students to fill in missing information about the glycolysis pathway and answer short questions about enzymatic deficiencies and how the utilization of glucose-6-phosphate is regulated.
The bc1 complex provides reduced cytochrome c to either the aa3 or cbb3 oxidases depending on oxygen conditions in R. sphaeroides. Strain BC-17 cannot photosynthesize due to lack of bc1 complex reducing cbb3. Some H217 mutations in the QI site can photosynthesize with DMSO but revert or are lethal without it. Future work should introduce H217 mutations into a DorR-/PpsR- background to uncouple effects on bc1 from changes to photosystem expression levels.
1) Rv3717 is a novel N-acetylmuramoyl-L-alanine amidase enzyme from Mycobacterium tuberculosis that was determined to have a single-domain structure at 1.7 Angstrom resolution.
2) Unlike other bacterial autolysins, Rv3717 lacks a separate cell-wall binding domain and instead uses its net positive charge for substrate binding.
3) The crystal structure revealed a flexible hairpin turn that partially occludes the active site, which may be involved in autoregulation of enzymatic activity similar to other bacterial amidases.
Austin Journal of Computational Biology and Bioinformatics is an open access, peer reviewed, scholarly journal dedicated to publish articles in all areas of research in Computational Biology and Bioinformatics.
The journal aims to promote research communications and provide a forum for researchers and other healthcare professionals to find most recent advances in the areas of Computational Biology and Bioinformatics.
Austin Journal of Computational Biology and Bioinformatics accepts original research articles, review articles, case reports and rapid communication on all the aspects of Computational Biology and Bioinformatics.
This document summarizes the rational design and generation of a catalytic antibody that selectively hydrolyzes a specific substrate. Researchers designed an antibody to bind a transition state analogue for the hydrolysis of a carbonate substrate. They generated monoclonal antibodies against a nitrophenyl phosphonate transition state analogue. One antibody was found to catalyze the hydrolysis of the carbonate substrate, displaying Michaelis-Menten kinetics. The antibody-catalyzed reaction had substrate specificity and was competitively inhibited by the corresponding phosphate transition state analogue, demonstrating the ability to rationally design catalytic antibodies.
This document summarizes research on the binding mechanism of Cu(II) ions to a poly-histidine peptide found in the venom of an African viper. The peptide contains 9 histidine residues and effectively binds copper, forming thermodynamically stable complexes. Experimental techniques like potentiometric titrations, UV-Vis, and EPR spectroscopy as well as molecular dynamics simulations were used to study the copper binding sites and structure of the complexes. The results show that copper prefers binding to histidine residues separated by one amino acid in the peptide and that more stable complexes form when the peptide adopts an alpha-helical structure.
This document reports on a study of the oxygenation properties of hemoglobin from the earthworm Lumbricus terrestris under varying conditions of pH, salts, and temperature. Key findings include:
1) Hemoglobin from L. terrestris exhibits relatively small cooperativity (free energy of 1.6-2.8 kcal/mol) and a large, pH-dependent Hill coefficient that reaches a maximum of 7.9.
2) Cations, not anions, control oxygen binding, with divalent cations having a larger effect than monovalent cations. Effectiveness decreases in the order Ca2+ > Sr2+ > Ba2+ and Mg2+ > Na+.
The document summarizes the complement system, which consists of over 20 proteins that work together to eliminate pathogens. It operates through three pathways - the classical, lectin, and alternative pathways. The classical pathway requires antibodies, while the alternative pathway can be activated independently of antibodies by pathogens. All three pathways involve a cascade of proteins that activate each other, ultimately forming the membrane attack complex that lyses the target cell membrane. The complement system also has roles beyond direct lysis, such as opsonization, inflammation, and activation of the immune response.
RNase contamination was preventing accurate measurement of RNA-protein binding between LARP6 and its RNA substrate. The researchers optimized native polyacrylamide gel electrophoresis with biotinylated RNA to visualize and quantify binding. They showed biotinylation worked without detecting RNase contamination. Continued gel condition optimization will improve resolution for determining the functional boundaries of the LARP6 La Module domain required for RNA binding.
This document summarizes a study that found neomycin, an aminoglycoside antibiotic, is capable of binding tightly to single-stranded poly(A) RNA with a Kd in the micromolar range. Circular dichroism experiments showed neomycin forms a complex with poly(A) and increases its melting temperature from 44°C to 61°C, suggesting neomycin strongly stabilizes the poly(A) duplex. Isothermal titration calorimetry found neomycin binds oligo(A)30 with a binding constant of 5.3×10^6 M^-1 and a stoichiometry of one neomycin per 10 adenine bases. This study demonstrates that neomycin can target single
Technological devices have become part of the everyday classroom. The teacher is able to use technology to help with interactive lessons as well as having access to online resources.
This document summarizes research on converting an engineered potassium-binding site in cytochrome c peroxidase (CCP) into a calcium-selective site through protein engineering and crystal structure analysis. The researchers previously engineered a potassium-binding site in CCP based on the structure of ascorbate peroxidase. They then designed mutants intended to bind calcium selectively instead. The crystal structure of the first mutant showed binding of a smaller cation like sodium rather than calcium due to disordering of a ligand. A second mutant was then designed and its crystal structure confirmed calcium binding with a fully coordinated ligand environment, demonstrating that an iterative engineering approach can switch cation selectivity in proteins.
Biomimetic Glycoside Hydrolysis by a Microgel Templated with a Competitive Gl...aaaa zzzz
This document summarizes the synthesis and characterization of biomimetic microgel catalysts templated with a competitive glycosidase inhibitor (galactonoamidine). The microgels were synthesized via free radical polymerization in the presence of a binuclear copper complex and the inhibitor. Computational modeling was used to determine the inhibitor binds to the copper complex through its amidine and hydroxyl groups. The resulting microgels were characterized and found to have a narrow size distribution and near-quantitative incorporation of the copper complex. The microgels show potential as biomimetic catalysts for glycoside hydrolysis.
Insertions and deletions are undervalued and underutilized source of diversity. Below is an example how a deletion can change metal selectivity of the CbiX chelatase.
The document summarizes characterization of the H134C mutant of the Thermus thermophilus Rieske protein. Key findings include:
1) The H134C mutant substitutes one ligating histidine for a cysteine, changing the iron-sulfur cluster ligation from 2Cys-2His to 3Cys-1His.
2) Studies using UV-visible spectroscopy, circular dichroism, and reaction with diethyl pyrocarbonate (DEPC) show the H134C mutant is stable across a wide pH range, unlike the similar mitochondrial protein mitoNEET.
3) Reaction of H134C with DEPC results in modification of the cluster but no reduction,
Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regul...Wei Fang
This document summarizes a study that used RNA sequencing to analyze changes in gene expression in the alga Chlamydomonas reinhardtii under different carbon dioxide (CO2) conditions and in a mutant lacking the CO2-concentrating mechanism regulator CIA5. The study found massive effects on the transcriptome, with almost 25% of genes affected. Distinct clusters of genes were identified that responded primarily to either CIA5 or CO2. Several clusters associated with CO2-concentrating mechanism genes were identified that may contain new candidate genes involved in inorganic carbon transport.
This document summarizes research exploring the biochemical properties and remediation applications of an unusual P450 system (XplA/B) found in Rhodococcus bacteria that is able to degrade the explosive compound RDX. Key findings include:
1) XplA has a high affinity for RDX (Kd = 58 μM) and XplB functions as its native reductase partner to efficiently degrade RDX.
2) Expression of both XplA and XplB in Arabidopsis plants enables significantly faster removal of RDX from contaminated liquid culture and soil compared to plants expressing only XplA.
3) Under anaerobic conditions, RDX degradation by XplA/B produces
1) Cryo-EM was used to determine the structure of the human nucleosome containing the histone variant H2A.B (H2A.B-NCP) at atomic resolution of 3.9 Angstroms.
2) The H2A.B-NCP structure is distinct from canonical nucleosomes, compacting around 103 base pairs of DNA in 1.2 superhelical turns compared to 145 base pairs in 1.7 turns for canonical nucleosomes.
3) A poly(ADP-ribose) polymerase 1 DNA binding domain was found to stabilize the dynamic H2A.B-NCP, allowing determination of its structure at higher resolution of 2.8 Ang
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
This document presents a structure/function model for the enzyme 1-pyrroline-5’ carboxylate reductase (P5CR) based on similarities to the β-hydroxyacid dehydrogenase enzyme family. The model is supported by evidence that recombinant E. coli P5CR has similar secondary structure to a β-hydroxyacid dehydrogenase and contains conserved functional domains. Site-directed mutagenesis of conserved residues in the proposed substrate-binding domain reduced catalytic efficiency. Chemical modification studies also provided insights into active site residues. The model proposes γ-glutamate semialdehyde as a potential true substrate based on inhibition studies.
2014. Pietkiewicz, Wahyudi. Synthesis of macrocycles that inhibit protein syn...Adrian Pietkiewicz
This document describes the synthesis of seven new analogues of the macrocyclic peptide sanguinamide B (SanB) and testing of their ability to inhibit protein synthesis in cancer cells. The analogues were designed by altering the amino acids at positions I and III of the SanB backbone, inverting stereochemistry at position III, and changing the protecting group on lysine. All analogues were tested for cytotoxicity against colon cancer cell lines and ability to inhibit protein synthesis. The lead compound with an IC50 of 15.9 μM against colon cancer cells contained an N6-carboxybenzyl-lysine at position I. This establishes the importance of this moiety for biological activity.
This document appears to be a midterm exam for a university course on biochemistry. It contains multiple choice and true/false questions about topics like glycolysis, gluconeogenesis, the citric acid cycle, and related metabolic pathways. It also asks students to fill in missing information about the glycolysis pathway and answer short questions about enzymatic deficiencies and how the utilization of glucose-6-phosphate is regulated.
The bc1 complex provides reduced cytochrome c to either the aa3 or cbb3 oxidases depending on oxygen conditions in R. sphaeroides. Strain BC-17 cannot photosynthesize due to lack of bc1 complex reducing cbb3. Some H217 mutations in the QI site can photosynthesize with DMSO but revert or are lethal without it. Future work should introduce H217 mutations into a DorR-/PpsR- background to uncouple effects on bc1 from changes to photosystem expression levels.
1) Rv3717 is a novel N-acetylmuramoyl-L-alanine amidase enzyme from Mycobacterium tuberculosis that was determined to have a single-domain structure at 1.7 Angstrom resolution.
2) Unlike other bacterial autolysins, Rv3717 lacks a separate cell-wall binding domain and instead uses its net positive charge for substrate binding.
3) The crystal structure revealed a flexible hairpin turn that partially occludes the active site, which may be involved in autoregulation of enzymatic activity similar to other bacterial amidases.
Austin Journal of Computational Biology and Bioinformatics is an open access, peer reviewed, scholarly journal dedicated to publish articles in all areas of research in Computational Biology and Bioinformatics.
The journal aims to promote research communications and provide a forum for researchers and other healthcare professionals to find most recent advances in the areas of Computational Biology and Bioinformatics.
Austin Journal of Computational Biology and Bioinformatics accepts original research articles, review articles, case reports and rapid communication on all the aspects of Computational Biology and Bioinformatics.
This document summarizes the rational design and generation of a catalytic antibody that selectively hydrolyzes a specific substrate. Researchers designed an antibody to bind a transition state analogue for the hydrolysis of a carbonate substrate. They generated monoclonal antibodies against a nitrophenyl phosphonate transition state analogue. One antibody was found to catalyze the hydrolysis of the carbonate substrate, displaying Michaelis-Menten kinetics. The antibody-catalyzed reaction had substrate specificity and was competitively inhibited by the corresponding phosphate transition state analogue, demonstrating the ability to rationally design catalytic antibodies.
This document summarizes research on the binding mechanism of Cu(II) ions to a poly-histidine peptide found in the venom of an African viper. The peptide contains 9 histidine residues and effectively binds copper, forming thermodynamically stable complexes. Experimental techniques like potentiometric titrations, UV-Vis, and EPR spectroscopy as well as molecular dynamics simulations were used to study the copper binding sites and structure of the complexes. The results show that copper prefers binding to histidine residues separated by one amino acid in the peptide and that more stable complexes form when the peptide adopts an alpha-helical structure.
This document reports on a study of the oxygenation properties of hemoglobin from the earthworm Lumbricus terrestris under varying conditions of pH, salts, and temperature. Key findings include:
1) Hemoglobin from L. terrestris exhibits relatively small cooperativity (free energy of 1.6-2.8 kcal/mol) and a large, pH-dependent Hill coefficient that reaches a maximum of 7.9.
2) Cations, not anions, control oxygen binding, with divalent cations having a larger effect than monovalent cations. Effectiveness decreases in the order Ca2+ > Sr2+ > Ba2+ and Mg2+ > Na+.
The document summarizes the complement system, which consists of over 20 proteins that work together to eliminate pathogens. It operates through three pathways - the classical, lectin, and alternative pathways. The classical pathway requires antibodies, while the alternative pathway can be activated independently of antibodies by pathogens. All three pathways involve a cascade of proteins that activate each other, ultimately forming the membrane attack complex that lyses the target cell membrane. The complement system also has roles beyond direct lysis, such as opsonization, inflammation, and activation of the immune response.
RNase contamination was preventing accurate measurement of RNA-protein binding between LARP6 and its RNA substrate. The researchers optimized native polyacrylamide gel electrophoresis with biotinylated RNA to visualize and quantify binding. They showed biotinylation worked without detecting RNase contamination. Continued gel condition optimization will improve resolution for determining the functional boundaries of the LARP6 La Module domain required for RNA binding.
This document summarizes a study that found neomycin, an aminoglycoside antibiotic, is capable of binding tightly to single-stranded poly(A) RNA with a Kd in the micromolar range. Circular dichroism experiments showed neomycin forms a complex with poly(A) and increases its melting temperature from 44°C to 61°C, suggesting neomycin strongly stabilizes the poly(A) duplex. Isothermal titration calorimetry found neomycin binds oligo(A)30 with a binding constant of 5.3×10^6 M^-1 and a stoichiometry of one neomycin per 10 adenine bases. This study demonstrates that neomycin can target single
Technological devices have become part of the everyday classroom. The teacher is able to use technology to help with interactive lessons as well as having access to online resources.
This resume summarizes Angela M. Rickelm's qualifications for a substance abuse counselor position. She has over 5 years of experience as a substance abuse counselor in Illinois and Texas, facilitating individual and group sessions, conducting assessments, and developing treatment plans. She holds a Bachelor's degree in Psychology and is a Certified Reciprocal AODA Counselor. She also has research experience in psychology and teaching experience as a teaching assistant.
Design of ultrasensitive DNA-based fluorescent pH sensitive nanodevices_MSsaheli halder
This document describes the design of new DNA-based fluorescent nanodevices that act as pH sensors with expanded sensitivity. The nanodevices use DNA i-motifs that undergo a conformational change between an i-motif structure at acidic pH and a duplex structure at basic pH. By incorporating chemically modified cytosines into the i-motif region, the researchers were able to tune the pH sensitivity range of the nanodevices. They created versions with brominated or methylated cytosines in different positions, and found that some achieved a collective sensing range from pH 5.3 to 7.5. The modified nanodevices also exhibited much larger signal changes between the two conformations, resulting in improved dynamic range for pH sensing.
A pH-independent DNA nanodevice for quantifying chloride transport in organel...saheli halder
This document describes a DNA nanodevice called Clensor that can precisely measure chloride ion concentration in organelles of living cells in a pH-independent manner. Clensor consists of sensing, normalizing, and targeting modules and uses a ratiometric fluorescence approach to quantify chloride levels across the full physiological range. The authors demonstrate that Clensor can be used to measure resting chloride concentration in lysosomes of Drosophila cells and show that lysosomal chloride is regulated by the intracellular chloride transporter DmClC-b.
This one sentence document does not provide enough context or information to create an accurate 3 sentence summary. The document contains only one word - "Lorem" - which is not meaningful on its own.
Recombinant antibody mediated delivery of organelle-specific DNA pH sensors a...saheli halder
This document describes the development of a new family of DNA pH sensors that can be targeted to specific intracellular locations using recombinant antibodies. The sensors incorporate a "handle" domain that binds recombinant antibodies identified through a phage display screen. One such antibody is fused to the membrane protein furin, allowing the sensors to be ferried along the furin endocytic pathway inside living cells. By changing the sequence of the pH sensitive domain, sensors were created that span pH ranges from 4 to 7.6. When targeted to cells expressing the antibody-furin chimera, the sensors map the spatiotemporal pH changes within intracellular compartments during furin trafficking. This targeting technique provides a generalizable strategy for delivering DNA nanode
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive function. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
La acción de tutela es un mecanismo de protección constitucional de los derechos fundamentales de los colombianos. Procede contra particulares cuando estos prestan un servicio público, afectan grave y directamente un interés colectivo, o cuando existe un estado de subordinación o indefensión hacia el afectado. También procede contra particulares que ejercen funciones públicas, pues en tal caso ostentan la calidad de autoridad pública.
This short document outlines two compositions by González Templos Bryan. It lists "Composition 1" and "Composition 2" but provides no further details about the pieces.
Este documento describe los primeros auxilios para heridas y quemaduras en la piel. Explica cómo clasificar heridas y quemaduras según su gravedad y profundidad, desde lesiones leves de primer grado hasta graves de tercer grado. Detalla los pasos básicos para el cuidado inicial de ambos tipos de lesiones, como limpiar y desinfectar la herida, enfriar una quemadura, aplicar apósitos estériles y buscar atención médica para las lesiones graves.
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Ben Kelty Summer Research Poster Presentation Benjamin Kelty
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Research Inventy : International Journal of Engineering and Scienceresearchinventy
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Proteins play key roles in living systems through catalysis, transport, and information transfer. They have a hierarchical structure including primary, secondary, tertiary, and quaternary levels. The primary structure is the amino acid sequence, and higher levels of organization are determined by the primary structure. Protein folding and interactions between residues determine the final 3D tertiary and quaternary structures, which are critical for protein function. Misfolded proteins can cause diseases.
Similar to tuning the pH Response of i-Motif DNA Oligonucleotides_Lannes_et_al-2015-ChemBioChem (20)
tuning the pH Response of i-Motif DNA Oligonucleotides_Lannes_et_al-2015-ChemBioChem
1. Tuning the pH Response of i-Motif DNA Oligonucleotides
Laurie Lannes,[a]
Saheli Halder,[b]
Yamuna Krishnan,[b, c]
and Harald Schwalbe*[a]
Introduction
In addition to the well-known double helix conformation, spe-
cific DNA sequences can form additional, more complex ter-
tiary structures stabilised by non-Watson–Crick base pairs.
Under specific conditions, cytosine- and guanine-rich sequen-
ces exhibit a rich polymorphism and can form quadruplex sec-
ondary structures known as i-motifs and G-quadruplexes.[1]
The i-motif structure consists of a tetraplex composed of
two anti-parallel duplexes connected by intercalated hemipro-
tonated cytidine·cytidine+
base pairs (C·C+
).[1a]
This pH-depen-
dent protonation of opposite cytidine base pairs can occur
under mild acidic conditions at the N3 position; the pKa of iso-
lated cytosine is 4.58.[2]
As a consequence, i-motif sequences
are fully folded within a pH range of 5–6.
The complementary strand can form G-quadruplexes that
are composed of four strands connected by planar G-tetrads
stacked on top of each other. Formation of G-tetrads relies on
Hoogsteen hydrogen bonds and is often dependent on the
presence of monovalent cations (Na+
, K+
, NH4
+
), occupying
the central channel between tetrads.[1b]
G-quadruplexes and i-motifs are present in tandem on com-
plementary strands in particular locations of the genome, in-
cluding telomeres,[3]
oncogene promoters,[4]
and centromeres.[5]
The colocalisation of these sequences has generated consider-
able interest in understanding their functions and whether
their functions might even be coupled.[6]
Direct evidence of the in vivo existence of i-motifs is still
missing. At first glance, the pH in the environment of the nu-
cleus should be too high for i-motifs to form. However, studies
showed that C-rich sequences can form at physiological pH in
a crowded environment[7]
or from a duplex under negative su-
perhelicity pressure.[8]
Several proteins have been identified to bind i-motif-compe-
tent sequences.[9]
For example, Hurley et al. recently discovered
the first protein (hnRNP L-like) that recognises and preferential-
ly binds to the i-motif conformation over random coil confor-
mations of bcl-2 C-rich promoter sequences (Py39wt).[9e]
In ad-
dition, by using ligands that have antagonist effects on i-motif
stability and subsequent binding to hnRNP L-like, they were
able to control bcl-2 expression in vitro.[9e,10]
In addition to their biological functions, the use of DNAs as
building blocks for nanodevices has become an attractive field
of research. In this field, C-rich DNAs have obtained considera-
ble attention due to their unique pH-switching capacity. In
2003, the Balasubramanian group designed the first i-motif-
based nanodevice by functionalising it at the 5’ and 3’ termini
with a fluorophore and a quencher, respectively. A switch in
pH allowed cyclic reversible generation of either an i-motif
(low pH) or a duplex (neutral pH).[11]
Protonation-dependent
transitions from duplex or random coil conformations to the i-
motif structure have since been implemented to design several
nanodevices. Applications are broad and as various as pH sen-
sors,[12]
logic gates,[13]
electronic components,[14]
nanopores for
substrate delivery,[15]
or ion nanochannels.[16]
Cellular pH sensors are particularly interesting nanoma-
chines, as the intracellular pH (pHi) has an important role in cel-
lular homeostasis. Cells do not maintain identical pH values
throughout, but each compartment has an optimum pH. For
instance, the nucleus and the cytosol have a pH of 7.2, where-
as mitochondria adopt a pH of 8.0, the Golgi a pH of 6.0–6.7
and the lysosomes a pH of 4.7.[17]
Acidification of the cell, for
example, is linked to apoptosis.[18]
Cancer cells undergo basifi-
cation (pHi >pH 7.4), which leads to a reversed pH gradient
between the intra- and extracellular environments.[18]
Hence,
Cytosine-rich single-stranded DNA oligonucleotides are able to
adopt an i-motif conformation, a four-stranded structure, near
a pH of 6. This unique pH-dependent conformational switch is
reversible and hence can be controlled by changing the pH.
Here, we show that the pH response range of the human telo-
meric i-motif can be shifted towards more basic pH values by
introducing 5-methylcytidines (5-MeC) and towards more
acidic pH values by introducing 5-bromocytidines (5-BrC). No
thermal destabilisation was observed in these chemically modi-
fied i-motif sequences. The time required to attain the new
conformation in response to sudden pH changes was slow for
all investigated sequences but was found to be ten times
faster in the 5-BrC derivative of the i-motif.
[a] L. Lannes, Prof. Dr. H. Schwalbe
Institute for Organic Chemistry and Chemical Biology
Center for Biomolecular Magnetic Resonance (BMRZ)
Johann Wolfgang Goethe-University Frankfurt
Max-von-Laue-Strasse 7, 60438 Frankfurt/Main (Germany)
E-mail: schwalbe@nmr.uni-frankfurt.de
[b] S. Halder, Dr. Y. Krishnan
National Centre for Biological Sciences, TIFR
GKVK Campus, Bellary Road, Bangalore 560065 (India)
[c] Dr. Y. Krishnan
Department of Chemistry, University of Chicago
E305, GCIS, 929 E, 57th Street, Chicago, IL 60637 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500182.
ChemBioChem 2015, 16, 1647 – 1656 2015 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim1647
Full PapersDOI: 10.1002/cbic.201500182
2. monitoring of the pHi is of high interest for diagnostics, drug
design, and better understanding of cellular processes.
In the case of i-motif-based pH sensors for in vivo applica-
tions, several issues drive the design of such switchable DNA
sequences. The device should respond in an adequate pH
range, according to the targeted cellular compartment. When
the organelle of interest undergoes a rapid change in pH, as
occurs in the endosome, the Golgi, or any organelle under pH
stress conditions, the pH sensor should also process a fast re-
sponse in order not to miss monitoring spatial and temporal
pH changes. Therefore, it is mandatory to investigate the pH
profile of various i-motifs in terms of the midpoint of titration
and the transition width of the titration, as well as the kinetics
of their folding.
In previous work, we characterised the pH-induced folding
pathway of the human telomeric i-motif DNA d[(CCCTAA)3CCC]
by static and time-resolved NMR spectroscopy.[19]
Our investi-
gations revealed a kinetic partitioning mechanism with a first
step in which two conformations (Scheme 1) are formed with
a rate constant on the order of 2 minÀ1
. Subsequent refolding
of the kinetically favoured conformation to the thermodynami-
cally more stable conformation was slow, with rate constants
on the order of 10À3
minÀ1
. At equilibrium, two distinct confor-
mations were populated at a ratio of 3:1. Cytosine-selective
isotope labelling schemes allowed us to assign both conform-
ers, which differ in the intercalation topology of the C·C+
base
pairs.[19–20]
The major conformer is closed by the C·C+
base pair
at the 5’-end position (5’E), whereas the minor conformer is
closed by the C·C+
base pair at the 3’-end position (3’E).
The human telomeric sequence was previously integrated
into a nanostructure to quantitatively assay the stability and
lifetime of various DNA nanostructures in vivo.[21]
The mutant
sequence I4, which presents an extra cytosine in each C-tract,
was implemented in an i-motif switch designed to probe the
pH evolution of endosomes in real time.[12d]
In this report, we investigated whether the pH response of
the human telomeric i-motif (I3) can be tuned by substituting
cytosines with 5-methylcytosine (5-MeC) and 5-bromocytosine
(5-BrC) or by elongating it with an additional cytosine (I4). This
approach is motivated by the different pKa(N3) values of free
5-MeC, 5-BrC, and C. Karino et al. determined that 5-MeC has
a pKa(N3) of 4.5, whereas C has a pKa(N3) of 4.4.[22]
Kulikowski
et al. found that 5-BrC has a pKa(N3) at 2.45, compared to 4.1
for C.[23]
Further, we investigated the influence of such modifi-
cations on the kinetics of i-motif formation at different pH
values.
Using various cytosine derivatives and extending the length
of the C·C+
strands allowed us to tune the pH range by ++0.14
and À0.22 pH units and the folding kinetics by a factor of 10,
whereas the previously observed partitioning of the folding
pathways remained unaltered.
Results and Discussion
We rationalised the positions of 5-MeCs and 5-BrCs according
to the structural organisation of the I3 i-motif.[19,24]
We decided
to position the modified cytosines in order to form homoge-
nous base pairing (i.e., 5-xC·5-xC+
, where x can be a methyl
group, a bromine substituent, or a hydrogen atom). Indeed,
we showed in previous work that the C·C+
imino proton is
dynamically bound to both cytidines across the strands, and
hydrogen bonding needs to be described by a double well po-
tential, which requires the pKa to be tuned on both sides of
the base pairing.[25]
Furthermore, we introduced predicted chemically modified
C·C+
base pairs in the middle of the C·C+
core, where the
modifications should lead to minimal interactions with loop
nucleotides.[19]
Scheme 1 presents the i-motif organisation of
the DNA sequences reported in Table 1. Further, C-rich oligonu-
cleotides presenting four Cn tracts (with n!2) are also expect-
ed to form an intramolecular i-motif.[6a,26]
i-Motif folding competence
In order to determine the stoichiometry of the i-motifs after
acidification, we carried out polyacrylamide gel electrophoresis
(PAGE). On denaturing PAGE, I3, I3Me4, and I3Br2 migrated in
an identical manner compared to a polydT sequence of identi-
cal number of nucleotides (polydT T21) (Figure 1A). Thus, intro-
duction of bromo- or methyl-substituted cytidines into the oli-
gonucleotides did not lead to any significant migration differ-
ence when DNA molecules were fully relaxed. As a conse-
quence, differences in migration on native PAGE can be inter-
preted as arising from differences in secondary structure. The
polydT sequences (dT10, dT21, and dT25) were used as size mark-
ers, assuming that their migration behaviour was not affected
by differences in pH. At pH 5.0 (Figure 1B), the sequences of
interest formed species that migrated roughly together with
T10, appearing twice as small as predicted from their actual
Scheme 1. Organisation of the 3’E and 5’E conformers of the human telo-
meric i-motif I3 (left) and its mutant I4 (right).[19]
The hemiprotonated cyto-
sine·cytosine+
(C·C+
) base pairs are depicted as full triangles. C2·C14+
and
C8·C20+
are composed of 5-methylcytosines, and C8·C20+
is composed of
5-bromocytosines in I3Me4 and I3Br2, respectively. Table 1. Oligonucleotide sequences.
Name Sequence 5’!3’ Name Sequence 5’!3’
I3 (CCCTAA)3CCC I4 (CCCCTAA)3CCCC
I3Me4 (C5m
CCTAA)3C5m
CC I3Br2 (C3TA2C5Br
CCTA2C3TA2C5Br
CC
ChemBioChem 2015, 16, 1647 – 1656 www.chembiochem.org 2015 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim1648
Full Papers
3. size. The intramolecular i-motif structure is more compact than
an ssDNA coil and is expected to migrate faster than dT21 se-
quences without structure. All sequences showed one strong
band, indicating that they formed a monomeric structure at
acidic pH. Interestingly, I3Br2 migrated slightly slower than I3
and I3Me4, possibly due to formation of a less compact struc-
ture. In both gels, the I3Br2 lane showed a higher light band
that might correspond to a stable dimer. Certain nucleic acid
secondary structures are not fully disrupted in regular denatur-
ing gel.[27]
We further tested i-motif formation by NMR spectroscopy.
The imino proton engaged in the C·C+
base pair has a charac-
teristic chemical shift around 15.5 ppm. Figure 2 shows the 1
H
1D spectra of each DNA sequence of interest at acidic pH, fo-
cusing on the 16–15 ppm region. In the corresponding NOESY
spectra, cross peaks could be observed between intercalated
C·C+
base pairs protons, caused by their close proximity (3.3 Š
average distance, as determined by NMR structure 1EL2;[24]
Fig-
ure S1 in the Supporting Information). This cross-peak pattern
provided additional evidence for i-motif formation. Notably,
the I3Br2 1D spectrum presented minor peaks around 14.5–
15 ppm that could belong to C·C+
imino protons from the
putative dimer form already observed by electrophoresis.[28]
We assessed the proportion of this species as ~5% at NMR
concentration. This marginal population was considered not
important in following experiments.
pH and thermal stability
We monitored pH-dependent i-motif formation by circular di-
chroism (CD) spectroscopy. The resulting CD spectra acquired
over the pH range 7.2 to 4.8 are presented in Figure 3. The
I3Me4 and I3Br2 sequences revealed similar spectral character-
istics to I3 and I4. At pH 5.0, the oligonucleotides (ODNs) dis-
played a maximum band around 288 nm and a minimum band
between 255 and 260 nm (individual values are given in
Table 2), in agreement with previous reports.[4b,d,g,29]
At pH 7.2,
the ODNs had a complete different profile, with a maximum
band near 275 nm and a minimum band near 250 nm, charac-
teristic of a single-stranded DNA random coil conformation.[30]
In addition, pH titration of the CD spectra of I3, I4, and I3Br2
revealed two distinct isoelliptic points, which represent strong
evidence for a transition between two discrete conformational
states.[31]
The pH-dependent CD spectra showed that the non-
natural nucleotides did not impair the formation of i-motif
structure. The introduction of 5-MeCs into i-motif sequences
has already been studied, and similar results as reported
herein have been observed.[24,32]
On the other hand, the intro-
duction of 5-BrCs was never reported.
Figure 1. A) 20% denaturing (8m urea) polyacrylamide gel (PAGE). The
polydT dT10, dT21, and dT25 were size marker oligonucleotides. B) 20% native
PAGE, buffered by TAE pH 5.0. Bands were visualised by UV shadowing.
Figure 2. A) Hemiprotonated cytidine·cytidine+
(C·C+
) base pairs with
a proton shared by both cytidines, as described by Lieblein et al. in 2012.[25]
B) C·C+
base pair imino proton region of 1D NMR spectra of i-motif DNA
sequences I3, I4, I3Me4, and I3Br2 at slightly acidic pH.
Table 2. Characteristics of the CD spectra of i-motif DNA sequences.
I3 I4 I3Me4 I3Br2
max. band[a]
[nm] 288.3 287.7 288.3 288.5
(275.4) (275.7) (274.5) (275.2)
min. band[b]
[nm] 257.3 260.3 254.7 254.5
(248.3) (247.1) (249.8) (248.3)
isoelliptic points 277.0 278.0 276.6 278.0
[Æ0.2 nm] 246.0 243.2 n.o. 244.2
[a] Average of values obtained in triplicate for 100% fraction folded/un-
folded. [b] Average of spectra measured in triplicate. n.o.: not observed.
Band values in brackets correspond to the unfolded state.
ChemBioChem 2015, 16, 1647 – 1656 www.chembiochem.org 2015 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim1649
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4. We chose the molar ellipticity at 288 nm for I3, I3Me4, and
I4 and 289 nm for I3Br2 as a reporter (ME288/289) for i-motif for-
mation to follow the state of folding for each of the sequences
over a pH range between 4.8 and 7.2. The pH-dependent fold-
ing was fully cooperative for all systems investigated. At the
lowest and highest pH values, we observed maximal and mini-
mal ME288/289, respectively. We conclude that the pH-induced
transition can be well titrated over the chosen pH range. Con-
sequently, we converted the ME288/289 into the DNA fraction
folded (FF). Plots of the FF against pH values are presented in
Figure 4.
In order to use an i-motif as a pH sensor, the midpoint of
the pH-dependent cooperative folding/unfolding transition
must coincide with the (cellular) pH of interest. We defined the
width of the pH transition as the pH response range of the C-
rich sequence. In order to compare the pH response ranges of
the various i-motifs studied, we defined this within an upper
and a lower pH limit as defined by 95% and 5% of the fraction
folded (FF95 and FF5, respectively; Table 3).
Considering the transitional pH (pHFF50) of each sequence,
we noticed that 5-BrC had the opposite effect of 5-MeC.
Indeed, I3Br2 revealed a DpHFF50 (pHFF50 (I3)–pHFF50 (I3Br2)) of
À0.33, whereas I3Me4 had a DpHFF50 of ++0.14. In comparison,
the elongation of the C-tracts in I4 showed a larger DpHFF50 for
++0.38 than for I3Me4. Further, by analysing the amplitude of
the pH response range of the sequences, differences in the co-
operativity of folding were ap-
parent. The transition range
(pHFF95–pHFF5) of I3 spanned
0.69 units. The pH response
range of I3Br2 was narrower,
with a transition range of 0.54,
contrary to I3Me4, which
showed a broader transition of
0.85. As a result, the introduction
of 5-MeCs led to a decrease in
cooperativity of the pH-induced
folding transitions, contrary to
what was observed upon intro-
duction of 5-BrCs.
From PAGE analysis, we dem-
onstrated that the investigated
sequences adopted an intramo-
lecular, monomeric i-motif. On
this basis, we calculated the
thermodynamic parameters from CD temperature denaturation
curves. We measured melting curves at two different pH
values. Here, it is relevant to compare the thermodynamic pa-
rameters of the sequences at a pH value within their pH re-
sponse region, leading to the same fraction of folded i-motif.
Consequently, we chose to measure melting curves at pHFF50.
In addition, we measured melting curves at pH 5.0. The result-
ing melting curves are presented in Figure 5.
As expected, the melting temperature (Tm) of each sequence
decreased as the pH increased (see Table 4). Based on the Tm
values at pH 5.0, I3Me4 was the most stable i-motif compared
to I3 and I3Br2, with the latter being the least stable. Interest-
ingly, at their respective pHFF50 values, these sequences had
similar Tm values.
Figure 3. CD spectra of i-motif-competent sequences I3, I3Me4, I3Br2, and I4 over the pH range 4.8–7.2. The
presented spectra were averaged over three successive acquisitions. The ellipticity was converted into molar
ellipticity.
Figure 4. pH melting curves of i-motif sequences I3, I3Me4, I3Br2, and I4
over the pH range 4.8–7.2 at 298 K. The plots are derived from molar elliptic-
ity at 288 nm (I3, I4, and I3Me4) or 289 nm (I3Br2), monitored during the pH
titration presented in Figure 3. The CD data were transformed into folded
fraction and plotted against pH values. Fitting was performed by using five
points measured in triplicate. The error bar dots were obtained by averaging
the values of the triplicate measures, the limits of the error bars correspond
to the highest and the lowest values.
Table 3. pH-response range of i-motif sequences.
95% folded 50% folded 5% folded
I3 5.90 6.26Æ0.01 6.59
I3Me4 5.88 6.40Æ0.02 6.73
I3Br2 5.68 5.93Æ0.01 6.22
I4 6.32 6.64Æ0.01 6.91
Æstandard error from fitting in Figure 4.
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5. At pH 5.0, all sequences were folded both at 25 and 378C
except I3Br2, of which 20% were in random coil DNA struc-
tures at 378C. At pHFF50, we observed reduced stability. Indeed,
at 258C, the i-motif populations of I3, I3Me4, and I3Br2 de-
creased by a third and almost completely disappeared at 378C.
I4 presented a higher stability, due to its extra two C·C+
base
pairs. Consequently, considering its pHFF50, the I4 i-motif at
258C was largely formed, but its folded conformation de-
creased by two-thirds at 378C. Nevertheless, for I4, the quadru-
plex form was still populated at its pHFF50, contrary to the
other sequences studied.
Recently, Xu et al. investigated the introduction of one or
two 5-MeCs at different positions into the I3 sequence. They
reported that the position of one modified cytosine showed
a limited influence on pHFF50. However, they observed a greater
effect on the pH response range when both cytosines of
a C·C+
base pair were substituted instead of one. This observa-
tion confirmed the relevance of our tuning approach discussed
above.[32c]
Interestingly, Bhavsar-Jog et al. reported a DpHFF50 of
++0.2 when the cytidine at position 4 in d[TTC3TAC4AC3TA2]
ODN was replaced by a 5-MeC, due to a decrease in coopera-
tivity.[33]
The introduction of only one 5-MeC into I3 leads to
a more modest DpHFF50,[32c]
and it is possible that different
parental sequences then undergo different effects due to the
modified cytidine introduction. In the case of I3Br2, it was strik-
ing that the exchange of only one C·C+
base pair by a homo
5-BrC·5-BrC+
base pair led to such tremendous effect on the I3
pH response range. The introduction of one 5-hydroxymethyl-
cytosine (5-hmC) in i-motif sequences also led to an acidic tu-
ning.[32c,33]
Yang et al. measured the base-pairing energies (BPEs) of 5-
xC·5-xC+
homodimers by guided ion beam tandem mass spec-
troscopy. They revealed that 5-MeC·5-MeC+
dimers comprised
a higher BPE than C·C+
dimers (177.4 and 169.9 kJmolÀ1
, re-
spectively).[34]
In line with these findings, we propose that 5-
MeC·5-MeC+
base pairs in i-motifs are more stable than C·C+
base pairs. As a result, the pH response range might be broad-
ened. Due to its lower pKa(N3), 5-BrC has a weaker proton
affinity than C. As a consequence, the 5-BrC·5-BrC+
base pair
might disrupt at more acidic pH than do C·C+
base pairs. How-
ever, 5-BrC·5-BrC+
dimers show only a slightly lower BPE
(168.5 kJmolÀ1
) than the unmodified parent, which implies
that both base pairs have similar stability.[34]
Kinetic investigations
In previous work, we elucidated the folding kinetics pathway
of the human telomeric sequence I3. We established that
1) the folding proceeds in two steps, and 2) two different
folded i-motif conformations are present at equilibrium. Initial-
ly, the less stable conformation is formed more rapidly, but at
equilibrium, two conformations are present: 5’E is three times
more populated than the 3’E conformer.[19]
Here, we first investigated whether this complex folding
pathway was conserved in the investigated i-motif sequences.
Thus, we performed time-resolved NMR spectroscopy to follow
the folding of the I4 sequence and used the NMR characteristic
chemical shift around 15.5 ppm arising from the proton shared
in the C·C+
base pairs.
After initiating folding by a temperature jump from 95 to
258C, we followed the evolution of successive 1D 1
H NMR
spectra over a period of 24 h. Figure 6A shows a sample of
spectra, focusing on the imino proton region, at different time
points. Eight peaks would be expected for each conformer;
however, due to overlap, only six apparent peaks were clearly
observed. We determined the intensity of each peak. These
data were then plotted against time to obtain the kinetic
traces presented in Figure 6B. Given the large effort required
to assign individual resonances for the two-state population of
i-motifs,[19–20,25,35]
we decided to focus here on analysis of the
kinetics without individual assignment, because the kinetic
process did not reveal any differences between different nucle-
otides in the sequence; in other words, there were no single
nucleotide-specific variations.
The four strongest peaks (at 15.77, 15.56, 15.48, and
15.41 ppm) showed a constant increase before reaching a pla-
teau. The peaks at 15.34 and 15.53 ppm showed an increase
during the first 30 min before decrease and finally reached
a plateau. The number of states involved in the kinetics of fold-
ing therefore remained unchanged compared to our previous
studies of the I3 sequence, allowing us to conclude that the
Figure 5. CD temperature melting curves of i-motif sequences I3, I3Me4,
I3Br2, and I4 at pH 5.0 (*) and at their transitional pH values (*) of 6.3 (I3),
6.6 (I4), 6.4 (I3Me4), and 5.9 (I3Br2). The molar ellipticities at 288 nm (I3,
I3Me4, I4Br2, and I4) or at 289 nm (I3Br2) were monitored and normalised to
be expressed as fraction folded.
Table 4. Melting temperatures (Tm) of the i-motif sequences at different
pH values, pH 5.0, and transitional pH values.
pH Tm
[a]
[8C] Tm
[b]
[C] pH Tm
[a]
[8C] Tm
[b]
[C]
I3 5.0 55.7 54.8Æ0.1 6.3 26.4 25.8Æ0.1
I3Me4 5.0 59.1 57.0Æ0.2 6.4 27.5 27.6Æ0.1
I3Br2 5.0 45.5 44.6Æ0.1 5.9 26.4 26.6Æ0.1
I4 5.0 68.4 67.9Æ0.2 6.6 35.1 34.1Æ0.1
[a] Median line method. [b] From fitting to f=y0 +a/1+exp((ÀxÀx0)/b), Æ
standard error.
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6. kinetics of I4 folding followed the same model as I3 (see Fig-
ure 6C).
We then studied folding of all sequences by CD spectrosco-
py to investigate folding kinetics in more detail. Depending on
the kinetics, i-motif folding was initiated by a pH jump using
an automatic mixing device (stopped-flow system) or by
manual mixing. We titrated folding kinetics over the pH ramp:
pHFF100, pHFF75, pHFF50, and pHFF25. Similar to the melting curves
described above, the characteristic i-motif CD signature at
288 nm was monitored over time after the pH jump. After
baseline and zero corrections, the molar ellipticity at 288 nm
was plotted against time to obtain the kinetic traces presented
in Figure S3. Most of the collected data could be fitted by
single or double exponential functions. Therefore, F-tests were
systematically run to compare the two possible fits for model
selection. According to these statistical tests, the best fitting
was always obtained by using a double exponential function.
The two rate constants describing the complex folding path-
way are given in Figure 7 and Table S5.
It is striking how the pH influences the folding kinetics of I3,
I3Me4, and I4. For these sequences, folding was greatly decel-
erated when we compared a protonation change at saturating
pH (~pH 8 to 5) to a change at non-saturating pH (~pH 8 to
pHFF75, for example), as previously observed for I3.[36]
In fact, it
took 0.5, 10, and 4 s for I3, I3Me4, and I4, respectively, to reach
the equilibrium plateau after a pH jump from 8 to 5, whereas
it took more than 1000 s after a smaller amplitude pH jump.
Interestingly, only I3Br2 did not show such strong effects. For
I3Br2 folding, equilibrium was reached in 100 s for a pH jump
towards saturating value; however, it took 150 and 200 s when
jumping to non-saturating pH values.
The folding rate constants k1 and k2 in Figure 7 reflected
these observations. When we compared the rate constants ob-
tained for the saturating pH jump and the non-saturating pH
jumps, we observed differences of a factor of 1000, 100, and
10000 for I3, I3Me4, and I4, respectively. It is noteworthy to
point out that both rate constants were affected in a similar
manner for the same pH jump. In contrast, rate constants
obtained at non-saturating pH jumps revealed no significant
differences.
Surprisingly, Chen et al., who studied pH-induced folding of
I3 by stopped-flow CD spectroscopy, reported that the single
exponential function was the best to describe their data.[36]
Ac-
cording to our observations, CD spectroscopy is a reliable
method to monitor the complexity of the i-motif folding mech-
anism, and all CD kinetic traces had to be fitted to a double
exponential function. Although CD spectroscopy was unable
to detect the different conformers 3’E and 5’E determined by
Figure 6. I4 folding kinetics investigation by time-resolved NMR. A) C·C+
base pairs imino proton region of NMR spectra recorded over 24 h. B) Imino proton
peaks intensities are plotted as a function of time to give kinetic traces. The grey dots correspond to the experimental data, and the orange dots correspond
to double exponential fitting f(t)=a”(1Àexp(Àk1 ”t))+c”(1Àexp(Àk2 ”t)). An arrow in the inserted spectra highlights the peak analysed. C) Model of the I4
folding pathway from the unfolded state (U) towards i-motif structures partitioned between two conformers, according to the model published by Lieblein
et al.[19]
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7. NMR spectroscopy, including single-labelled nucleotides,[19]
be-
cause their optical signatures are equivalent, the partitioning
of the folding pathway between the two conformers can nev-
ertheless be determined unambiguously and characterised by
two constant rates.
According to our data for I3, I3Me4, and I4, both folding
steps were influenced by the proton concentration or, more
precisely, by a pH range, described as protonation-saturating
and -non-saturating. Only the I3Br2 case was different because
only the first rate constant (k1) showed this behaviour. It ap-
peared clear that the first conversion from a random coil DNA
conformation to the i-motif, with preferred formation of the
less stable 3’E conformer, revealed a pH dependence. However,
the second step involved a conversion from the 3’E conformer
to 5’E conformer. The observation of a pH dependence for this
second step, which involves structural rearrangement between
two folded conformations, suggests that C·C+
base pairs need
to be deprotonated to be disrupted, and reprotonation is re-
quired for the formation of the compact i-motif. This deproto-
nation/reprotonation step, in turn, leads us to propose that un-
folded or partially unfolded intermediates need to be involved
in structural conversion. Interestingly, I3 and I4 showed similar
k1 rate constants, but the k2 constant rate for I4 was tenfold
smaller than for I3. This finding suggested that only the
second step, but not the first folding step, was affected by the
number of C·C+
base pairs to be formed. This finding suggests
that formation of the base pairs in the first step is simultane-
ous, and that conversion of the two conformers is influenced
by the extra two C·C+
base pairs.
Conclusion
Because of the slightly higher and significantly lower pKa
values of the N3 atoms of 5-MeC and 5-BrC, respectively, we
were able to tune the pH response of i-motif DNA oligonucleo-
tides. NMR and CD spectroscopy showed that the chemical
modifications do not prevent the studied DNAs from forming
an i-motif at slightly acidic pH values. Gel electrophoresis re-
ported the formation of only intramolecular folding. The new
sequences containing 5-MeCs and 5-BrCs displayed a coopera-
tive pH response. This behaviour makes them suitable for im-
plementation in nanodevices. Introduction of 5-MeCs in I3Me4
decreases the cooperativity of folding and therefore broadens
the pH response range, especially toward more basic values,
which could make I3Me4 suitable for monitoring the Golgi net-
work pH between 6 and 6.7 (I3Me4 responds over a pH range
of 5.88–6.73).[17]
Elongation of the C-tracts is also an interesting
strategy, which was already explored,[12a]
to tune the response
toward more basic values. I4 can monitor a more basic pH
than I3Me4, but once the pH response range is shifted, then
the acidic range detectable by I3 is lost with I4. On the contra-
ry, the introduction of 5-BrCs in I3Br2 leads to the opposite
effect: the pH response range is shifted towards more acidic
values.
We did not observe thermal destabilising effects, due to the
introduction of modified cytosine residues. Nevertheless, we
found out that I3, I3Me4, and I3Br2 i-motifs are poorly populat-
ed at 378C at their respective transitional pH values, which
makes them suboptimal for applications at physiological tem-
perature. Thus, I4 sequences present an advantage, because
they still show a large i-motif population at 378C.
Figure 7. Folding rate constants k1 and k2 of I3, I3Me4, I3Br2, and I4, corre-
sponding to different pH jumps. The folding of the DNA sequences was trig-
gered by a pH jump from pH 8 to acidic pH values by using stopped-flow
mixing or manual mixing. The folding was then monitored by circular di-
chroism at 288 nm. The kinetics were titrated over a pH ramp composed of
one protonation-saturating pH jump (pH 8 to 4.89 or 4.96) and three proto-
nation-non-saturating pH jumps. The rate constants k1 and k2 were obtained
by fitting the kinetic traces of Figure S3 to a double exponential function.
The average of the k2 values for the non-saturating pH jumps of I3, I4, and
I3Me4 and the average of all k2 values of I3Br2 were plotted; error bars cor-
respond to the maximum and the minimum values. *Standard error from fit-
ting. **Maximum and minimum k2 values.
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8. Finally, we demonstrated that the partitioned folding path-
way of the i-motif is conserved for all studied C-rich sequences.
The introduction of 5-MeC does not change the folding kinetic
rate, compared to I3. On the contrary, 5-BrC accelerates the
folding kinetics. Elongation of the C-tracts leads to a decrease
in the folding rate, due to a much slower conformer conver-
sion during the second step of the folding mechanism. As a
consequence, I4 needs hours to reach equilibrium.
I3 analogue I3Me4 showed that the introduction of 5-MeC
slightly decreased the cooperativity of folding and broadened
the pH response range toward more basic values (++0.19). We
could imagine that the introduction of 5-MeCs in I4 would pro-
duce a similar effect. Knowing that I4 has a pH response range
between 6.3 and 6.9, the addition of 5-MeCs might lead to the
design of DNA sequences that could monitor neutral and
slightly basic pH conditions. Similarly, we could transfer to 5-
BrC-I4 analogues the properties observed for I3Br2. We
showed that 5-BrC introduction shifted the pH response range
of the parental sequence towards more acidic values and ac-
celerated the folding kinetics. The resulting pH response range
of a 5-BrC-I4 analogue might overlay the I3-responsive pH
range. This I4 analogue would have an advantage over I3 in
that it would have thermal stability. In addition, we showed
that 5-BrCs could accelerate I4 folding kinetics.
The tuning strategies present advantages and disadvantages
on different levels which inevitably require compromises if ap-
plied to nanodevices. The comprehensive biophysical analysis
presented here shows that it is paramount to perform a com-
plete analysis with thermal stability and kinetic investigations
of i-motif sequences for optimisation in their application as cel-
lular nanodevices.
We decided to target C·C+
base pairs in our tuning tactics,
but the analysis of i-motif sequences found in promotor se-
quences, as in bcl-2 or c-myc, suggests that long loops induce
a pH stabilisation effect.[29]
The loops are suspected to have
a capping effect. The design of the loops in order to optimise
the formation of stacking of loop nucleotides with a C·C+
-cyti-
dine core, by using Watson–Crick (WC) or non-WC base pairing
into the loops, represents an interesting direction to follow.
The 5’- and 3’-end regions could also be exploited to introduce
supplementary stabilising elements. For instance, Nesterova
et al. shifted the transitional pH of the ODN d[(C5T3)3C5] from
6.9 to 7.2 by introducing a C-rich sequence in the loop of
a hairpin, with the stem of the hairpin leading to the stabilisa-
tion of the i-motif with regard to pH and temperature.[37]
In summary, the biophysical optimisation of the pH response
for various i-motifs, using natural and non-natural cytosine de-
rivatives with regard to stability and folding kinetics, will pro-
vide key insights for applications in bio-nanotechnology and
beyond for optimising sequence–response relationships for
this exciting class of tuneable oligonucleotides.
Experimental Section
DNA oligonucleotides: Oligonucleotides were purchased from Eu-
rofins MWG Operon. After HPLC purification, DNA samples were
freeze-dried and desalted by using microconcentrators with
a 3 kDa cut-off (Vivaspin 2, Sartorius). For the I4 ODN, a LiClO4/ace-
tone precipitation was performed to replace DNA counterions
from the HPLC step with lithium, and the ODN was then dissolved
in water. DNA concentrations were determined by UV/Vis spectros-
copy on a Cary50 UV-spectrophotometer by using extinction coeffi-
cients at 260 nm (e260), as presented in Table 5.
Sample preparation: Samples for circular dichroism (CD) spectros-
copy experiments had DNA concentrations of 19.5–20 mm for I3, I4,
I3Me4, and I3Br2, and all samples were prepared from the same
stock solution. The oligonucleotides were buffered by 25 mm po-
tassium acetate buffer over the pH range 4.8–5.6, or by 25 mm po-
tassium phosphate buffer over a pH range of 5.8–7.2. The samples
were incubated at 958C for 10 min and left at 48C at least one day
for equilibration before measurement.
Samples for static NMR spectroscopy were systematically snap-
cooled before measurement. DNA concentrations were 2 mm (I3)
600 mm (I4), 150 mm (I3Br2), and 70 mm (I3Me4). The samples were
buffered by 25 mm potassium phosphate buffer at pH 5.3 (I3 and
I3Br2) or 5.5 (I4 and I3Me4). The samples were supplemented with
10% of D2O, and DSS was used as an internal reference.
The I4 NMR sample for kinetics measurement was composed of
300 mm I4 DNA, 25 mm potassium phosphate buffer (pH 6.4), and
10% D2O; DSS was used as an internal reference. The sample was
incubated 5 min at 958C immediately prior to acquisition.
Denaturing PAGE: 20% polyacrylamide gel was mixed with 1”
TBE buffer pH 8.3 and urea (8m). I3, I4, I3Me4, I3Br2, T10, T21, and
T25 (150 mm each) were combined with loading buffer (99% forma-
mide, 0.01% bromophenol) and loaded on the gel. The gel was
run at room temperature in 1” TBE buffer with a current of 220 V
applied. Band migration was revealed on a silica gel plate under
UV light shadowing and photographed with a digital camera.
Native PAGE: 20% polyacrylamide gel was prepared by using 1”
TAE buffer, pH 5.0, 48C. DNA samples were combined with loading
buffer (50% glycerol, 5” TAE buffer, pH 5.0, 48C). All samples were
incubated at 958C for 10 min and stored overnight at 48C before
loading. The gel was run at 48C in 1” TAE buffer with a current of
60 V applied. DNA bands were visualised on a silica gel plate under
UV light, and the gel was then photographed with a digital
camera.
CD spectroscopy methods: The CD spectra and temperature melt-
ing curves were recorded on a Jasco J-810/815 CD spectropolarim-
eter equipped with a Jasco PTC-4235L Peltier thermostated cell
holder. The cell chamber was flushed with a constant nitrogen
flow to avoid water condensation on the measurement cuvette.
Table 5. Oligonucleotide sequences and their extinction coefficients
(e260).
Name Sequence 5’!3’ e260 [mcmÀ1
]
I3 (CCCTAA)3CCC 185900[a]
I4 (CCCCTAA)3CCCC 214700[a]
I3Me4 C5m
CCTA2C5m
CCTA2C5m
CCTA2C5m
CC 180450[b]
I3Br2 C3TA2C5Br
CCTA2C3TA2C5Br
CC 178830[b]
[a] Calculated by using the nearest-neighbour model. [b] Calculated by
using the base composition method. For 5-MeC and 5-BrC, e260 values of
5.7 and 3.1 mLmmolÀ1
, respectively, were used.
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9. For all measurements, a CD quartz cuvette with a path length of
1 mm was used.
The CD spectra were recorded at 258C over a spectral window of
220–330 nm, with data sampling of 0.2 nm at a scan speed of
50 nmminÀ1
. Spectra were the result of the average of three suc-
cessive acquisitions. A baseline correction was applied by using an
adequate buffer solution. Only the points at critical pH values (that
is, plateaus, inflection points, and transitional points) were mea-
sured in triplicate on independent samples to obtain error bars.
The ellipticity values were transformed into molar ellipticity, ac-
cording to Equation (1):
½qŠ ¼ ðq  MÞ=ðc  l  10Þ ð1Þ
where [q] is the molar ellipticity [degcm2
dmolÀ1
], q is the ellipticity
[mdeg], M the molecular weight [gmolÀ1
], l is the path length
[cm], and c is the concentration of the sample in [gmLÀ1
]. The
molar ellipticity values at 288 nm (I3, I3Me4, and I4) or 289 nm
(I3Br2) for each pH point were extracted and normalised to be
expressed as the fraction folded (1 or FF). Data for the triplicate
points were fitted with a three-parameter sigmoidal function: f=a/
(1+exp(À(xÀx0)/b)) by using SigmaPlot 12.5 software. x0 corre-
sponds to the transitional pH.
Temperature denaturing curves were obtained by monitoring the
CD at 288 or 289 nm along a temperature gradient of 4–958C, at
a rate of 0.58CminÀ1
. One data point was recorded every 0.58C.
The measured data were normalised and expressed as fraction
folded. We determined the melting temperature by the median
line method[38]
and by fitting.
Stopped-flow CD: SFCD measurements were carried out on
a Pistar-180 system (Applied Photophysics) set up for CD detection.
DNA sample solutions (10 mm DNA in buffer A: 45 mm KCl, 2.5 mm
K2PO4, pH 8.02) were rapidly mixed with buffer solution (buffer B:
25 mmK2HPO4 at different pH values) in a 1:1 ratio. The mixture
creates a pH jump in the direct environment of DNA molecules
from basic to acidic conditions. All acquisitions were performed at
258C through a path length set at 10 mm. The K+
cation concen-
tration was kept constant before and after mixing (50 mm). CD
evolution was monitored as function of time at 288 nm with
a bandwidth of 8 nm. Kinetics traces were recorded over different
periods, according to the ODN and the conditions (2–1000 s). Ten
thousand points were recorded for each trace, independent of the
acquisition time. Each condition was recorded five times (for traces
!200 s) or ten times (for traces 200 s) and then averaged. A t0
point was recorded for each condition by mixing DNA solution
against buffer A. Baseline corrections (buffer A against buffer A,
and buffer A against buffer B) were performed for each condition
and the t0 point, according to the same parameters and repetition
number of the corresponding pH jump experiment. The ellipticity
was baseline and zero corrected before being converted into
molar ellipticity (ME), as defined previously. In order to improve
the signal/noise ratio, we averaged the ME of five successive time
points, (except for I3 kinetics at pHFF75 and pHFF50) and plotted the
result against time.
The kinetics of I4 at pHFF75, pHFF50, and pHFF25 were recorded on the
same “normal” CD spectropolarimeter as describe above. The
mixing was manually performed, which led to a dead time of 15 s
before the beginning of the measure. The temperature was set at
258C, and the path length was 10 mm. A full spectrum (220–
330 nm) was recorded every 2 min with data sampling at 0.5 nm,
at a scan speed of 100 nmminÀ1
, over about 120 min, which repre-
sented 61 spectra in total. The same baselines and t0 point were re-
corded as described for stopped-flow CD kinetics measures. Each
kinetics experiment was performed once. After baseline and zero
corrections, the ellipticity was converted into ME. The ME values at
287.5, 288.0, and 288.5 nm were averaged and plotted against
time.
Traces presented in Figure S4 were fitted to a 4-parameter or 5-
parameter double exponential function respectively, in SigmaPlot
12.5:
MEðtÞ ¼ a  ð1ÀexpðÀb  tÞÞ þ c  ð1ÀexpðÀd  tÞ
and
MEðtÞ ¼ ME0 þ a  ð1ÀexpðÀb  tÞÞ þ c  ð1ÀexpðÀd  tÞ
An F-test was systematically run to define the best fitting function.
Static NMR spectroscopy: Spectra were recorded on a Bruker
600 MHz (I3, I3Br2, and I3Me4) or a 950 MHz (I4) spectrometer
equipped with a cryogenic probe at 298 or 288 K (I3). The 1
H 1D
spectra used jump-and-return for water suppression,[39]
with a
jump-and-return delay set at 30 ms (I3), 37 ms (I3Me4), 38 ms (I3Br2),
and 15 ms (I4).
Time-resolved NMR spectroscopy: Folding kinetics were moni-
tored by real-time NMR spectroscopy on a Bruker 600 MHz spec-
trometer equipped with a cryogenic probe at 298 K. The sample
was incubated for 5 min at 958C, just before acquisition of a
pseudo-2D experiment with a jump-and-return sequence for water
suppression.[39]
This pulse sequence recorded successive 1D 1
H
spectra at several time points. The jump-and-return delay time was
set to 25 ms, the carrier frequency in the proton dimension was set
to the water frequency, and the repetition delay was 1 s. A total of
16384 1D spectra were recorded with 5.3 s per spectrum, corre-
sponding to an accumulation of four scans. After the five denatur-
ing minutes and the first spectral acquisition, 6.5 min elapsed.
Kinetic data were processed with TopSpin 3.2 (Bruker Biospin).
Kinetics data were fitted to a double exponential function with
SigmaPlot 12.5:
Iimino peak ¼ a  ð1ÀexpðÀb  tÞÞ þ c  ð1ÀexpðÀd  tÞÞ
where Iimino peaks corresponded to the extracted intensity of imino
peaks, and the coefficients b and d corresponded to two rate con-
stants (k1 and k2) describing kinetic partitioning.[19]
Acknowledgements
The authors thank Dr. Boris Fürtig and Irene Bessi for insightful
discussion and Dr. Alexey Cherepanov for stopped-flow CD sup-
port. H.S. is member of the DFG-funded cluster of excellence:
macromolecular complexes. BMRZ is supported by the state of
Hessen.
Keywords: bromocytidine · cytosine-rich DNA · i-motifs ·
methylcytidine · nanodevices · pH sensors · telomeric DNA
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Manuscript received: April 8, 2015
Accepted article published: May 28, 2015
Final article published: June 30, 2015
ChemBioChem 2015, 16, 1647 – 1656 www.chembiochem.org 2015 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim1656
Full Papers