Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC₅) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.     

Crystal structure and chirality of natural floridoside

The crystal structure and absolute configuration of natural floridoside (2-O-alpha-D-galactopyranosylglycerol) were determined by single-crystal X-ray diffraction analysis. The space group is orthorhombic P2(1)2(1)2(1) with Z=4, a=4.885(1), b=9.734(1), c=23.886(2) A at 296 +/- 2 K. The structure was solved by a direct method and refined to R=0.0351 from 1914 reflections of Cu Kalpha radiation.     

The influence of oligodeoxyribonucleotide phosphorothioate pyrimidine strands on triplex stability.

The analogues of the homopyrimidine oligonucleotide dT15 has been synthesized. The analogues, contains phosphorothioate bonds of a mixture of diastereoisomers or one of the two stereoisomer (either Rp or Sp). The analogues were mixed under conditions conductive to the formation of triple stranded assemblies. The mixtures were characterized by their thermal stabilities (Tm values) and CD spectra.     

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.     

Effect of histone composition on the stability of chromatin structure

The mode of fragmentation of chromatin by micrococcal nuclease has been studied in nuclei from different sources at physiological ionic strength and low temperature. During digestion, the size of chromatin was reduced until an average S value of 95–100 (hen erythrocyte) or 60–65 (rat liver) was attained. The accumulation of these structures correlated with the period of maximum solubility (80%), indicating that the bulk of chromatin behaved in this manner. Further digestion did not result in a corresponding decrease in S value but in a bimodal sedimentation pattern. As opposed to this behavior, chromatin containing actively acetylated core histones showed a continuous variation in size during the digestion. Indirect immunoprecipitation of chromatin by anti-H5 antibody and sheep anti-rabbit antibody revealed that the acetylated chromatin is partially depleted of H5.     

Selectivity of polyamines on the stability of RNA-DNA hybrids containing phosphodiester and phosphorothioate oligodeoxyribonucleotides.

RNA-DNA hybrid stabilization is an important factor in the efficacy of oligonucleotide-based antisense gene therapy. We studied the ability of natural polyamines, putrescine, spermidine, and spermine, and a series of their structural analogues to stabilize RNA-DNA hybrids using melting temperature (Tm) measurements, circular dichroism (CD) spectroscopy, and the ethidium bromide (EB) displacement assay. Phosphodiester (PO) and phosphorothioate (PS) oligodeoxyribonucleotides (ODNs) (21-mer) targeted to the initiation codon region of c-myc mRNA and the corresponding complementary RNA oligomer were used for this study. In the absence of polyamines, the Tm values of RNA-PODNA and RNA-PSDNA helices were 41 +/- 1 and 35 +/- 1 degrees C, respectively, in 10 mM sodium cacodylate buffer. In the presence of a hexamine analogue of spermine at a concentration of 25 microM, the hybrids were stabilized with Tm values of 80 and 78 degrees C, for RNA-PODNA and RNA-PSDNA, respectively. The d(Tm)/d(log[polyamine]) values, representing the concentration-dependent stabilization of hybrid helices by polyamines, increased from 10 to 24 for both the RNA-PODNA and RNA-PSDNA helices. Bisethyl substitution of the primary amino groups of the polyamines reduced the hybrid stabilizing potential of the polyamines. Among the homologues of spermidine [H2N(CH2)3NH(CH2)nNH2, where n = 2-8; n = 4 for spermidine] and spermine [H)N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-8; n = 4 for spermine], spermidine and spermine were the most effective agents for stabilizing the hybrid helices. At a physiologically compatible concentration of 150 mM NaCl, the hybrid helix formed from PODNA was more stable than that formed from PSDNA in the presence of polyamines. CD spectroscopic studies showed that the hybrids were stabilized in a conformation close to A-DNA in the presence of polyamines. The relative binding affinity of the polyamine homologues for the hybrid helices, as measured by the EB displacement assay, followed the same order in which they stabilized the hybrids. These results are important in the antisense context and in the general context of polyamine-nucleic acid interactions, and suggest that pentamine and hexamine analogues of spermine might be useful in improving the efficacy of therapeutic ODNs.     

Effect of oxidative stress on stability and structure of neurofilament proteins.

Neurofilament proteins are highly phosphorylated molecules in the axonal compartment of the adult nervous system. We report the structural analysis of neurofilament proteins after oxidative damage. SDS-PAGE, immunoblotting, circular dichroism, and Fourier transform infrared spectroscopy were used to investigate the relative sensitivity of neurofilaments to oxidative stress and to identify changes in their molecular organization. An ascorbate-Fe+3-O2 buffer system as well as catechols were used to generate free radicals on a substrate of phosphorylated and dephosphorylated neurofilaments. By Fourier Transform Infrared spectroscopy and circular dichroism, we established that the neurofilament secondary structure is mainly composed of alpha-helices and that after free radical damage of the peptide backbone of neurofilaments, those helices are partly modified into beta-sheet and random coil structures. These characteristic reorganizations of the neurofilament structure after oxidative exposure suggest that free radical activity might play an important role in the biogenesis of the cytoplasmic inclusions found in several neurodegenerative diseases.     

Effect of Ficoll 70 on thermal stability and structure of creatine kinase

The effect of Ficoll 70 on the thermal stability and structure of creatine kinase (CK) was studied using far-UV CD spectra and intrinsic fluorescence spectra. The thermal transition curves monitored by CD spectra were fitted to a two-state model using a modified form of the van’t Hoff equation to obtain the transition temperature (T _m) and enthalpy change (ΔH _u) of thermally induced denaturation of CK in the absence and presence of Ficoll 70. An increase in T _m with constant ΔH _u was observed with increasing Ficoll 70 concentration, suggesting that Ficoll 70 enhances the thermal stability of CK. Fluorescence spectral measurements confirmed this protective effect of Ficoll 70 on CK structure. In addition, we observed a crowding-induced compaction effect on the structure of both native state and thermally denatured state of CK in the presence of Ficoll 70, which is more obvious on the structure of the denatured ensemble compared to that of the native ensemble. Our observations qualitatively accord with the predictions of previously proposed crowding theory for the effect of intermolecular excluded volume on protein stability and structure. These findings imply that the effects of macromolecular crowding are essential to our understanding of protein folding and unfolding occurring in vivo.     

Crystal violet as an i-motif structure probe for reversible and label-free pH-driven electrochemical switch

A simple pH-induced electrochemical switch based on an i-motif structure is developed by using crystal violet as a selective electrochemical probe for the i-motif structure. Thiol-modified cytosine-rich single-strand oligonucleotide (C-rich ssDNA) can be self-assembled on the gold electrode surface via gold–sulfur interaction. Crystal violet is employed as an electrochemical probe for the i-motif structure because of its capability of binding with the i-motif structure through an end-stacking mode. In acidic aqueous solution, crystal violet may approach the electrode surface owing to the formation of the i-motif structure, resulting in an obvious signal, so-called “ON” state. Whereas in neutral or basic aqueous solution, the i-motif structure unfolds to dissociative single strand, which causes crystal violet to leave from the electrode surface, and a weak signal is obtained, so-called “OFF” state. In addition, in the range of pH 4.6–7.3, the increase in current has a good linear relationship (R = 0.989) with pH value in the testing solutions. This pH-driven electrochemical switch has the advantages of simplicity, sensitivity, high selectivity, and good reversibility. Furthermore, it provides a possible platform for pH measurement.     

Effect of phospholipid structure on stability and survival times of liposomes in circulation

The phosphatidylcholine (PC) component of liposomes was structurally modified by replacing its C-1, or both C-1 and C-2, ester linkage(s) with an ether and/or carbamyl bond(s) or by changing its steric configuration. Small unilamellar liposomes were formed from PC, traces of the corresponding 14C-labeled PC and cholesterol in the presence of 6-carboxyfluorescein (02.M) by sonication, and purified by centrifugation. These liposomes were administered intravenously to rats, and their stability in blood as well as the rate of their clearance from the circulation were determined. Stability and survival times of liposomes were markedly increased by modifying both the C-1 and the C-2 ester linkages in PC. A similar but quantitatively smaller effect was observed when only the C-1 ester linkage was modified. However, the stability remained unaffected by changing the steric configuration of PC, but this modification influenced the clearance rate of liposomes from the circulation. These results demonstrate that both stability in blood and the clearance rate from circulation can be modulated by structurally modifying the ester linkages in the phospholipid component of liposomes.     

Effect of polymyxin B on the structure and the stability of lipid layers

Summary Polymyxin B (PX) does not penetrate phospholipid monolayers and bilayers at low field strength across the lipid layers. The degree of penetration of PX is evaluated from its effect on the capacitance of the monolayers and on the conductance of the bilayers. PX added to one side of a bilayer causes its destabilization, it also enhances destabilization of lipid monolayers at positive electric fields across the surface layer in the direction of the adsorbed PX. PX lowers very little the fluorescence polarization of 1,6-diphenyl 1,3,5 hexatriene embedded in phospholipid vesicles. It is suggested that the penetration mechanism of PX into gram-negative bacteria is based on transient local breakdown of the plasma membrane.     

Effect of phosphatidylcholine vesicle size on chirality induction and chiral discrimination

The effect of the size of phosphatidylcholine (PC) vesicles on the induction of chirality and chiral discrimination was examined. Three kinds of vesicles formed with l-dimyristoyl, l-dipalmitoyl, or egg yolk PCs induced circular dichroisms (CDs) with the sign and intensity of the Cotton effect different from those of monomeric PCs. The CD intensity of the vesicles increased with a decrease in the vesicle size. Furthermore, the helicity of heterohelicene derivatives in a rapid equilibrium between right-handed (P) and left-handed (M) enantiomers was biased toward the M enantiomer side in l-PC vesicles, implying chiral discrimination by the vesicles. The extent of the bias toward the M enantiomer increased with an increase in vesicle size. Both the chirality induction and chiral discrimination were enhanced in a low-fluidity gel phase in comparison with those in a high-fluidity liquid-crystalline phase for every kind of vesicle of every size examined.     

Effect of conserved intersubunit amino acid substitutions on Hfq protein structure and stability

Hfq is a thermostable RNA-binding bacterial protein that forms a uniquely shaped homohexamer. Based on sequence and structural similarity, Hfq belongs to the like-Sm (LSm) protein family. In spite of a rather high degree of homology between archaeal and eukaryotic LSm proteins, their quaternary structure is different, usually consisting of five to eight monomers. In this work, the importance of conserved intersubunit hydrogen bonds for the Hfq spatial organization was tested. The structures and stabilities for the Gln8Ala, Asn28Ala, Asp40Ala, and Tyr55Ala Hfq mutants were determined. All these proteins have the same hexamer organization, but their stability is different. Elimination of a single intersubunit hydrogen bond due to Gln8Ala, Asp40Ala, and Tyr55Ala substitutions results in decreased stability of the Hfq hexamer. Tyr55Ala Hfq as well as the earlier studied His57Ala Hfq has reduced protein thermostability, which seems to correspond to an opening of the protein hydrophobic core.     

The formation pathway of i-motif tetramers

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C•C⁺ pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TC_n]₄ tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TC_n association results in the formation of several tetramers differing by the number of intercalated C•C⁺ pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C•C⁺ pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power −2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting.     

pH-Dependant fluorescence switching of an i-motif structure incorporating an isomeric azobenzene/pyrene fluorophore

In this study, we synthesized an Azo-py phosphoramidite, featuring azobenzene and pyrene units, as a novel fluorescent and isomeric (trans- and cis-azobenzene units) material, which we incorporated in an i-motif DNA sequence. We then monitored the structural dynamics and changes in fluorescence as the modified DNA sequences transformed from single strands at pH 7 to i-motif quadruplex structures at pH 3. After incorporating Azo-py into the 4A loop position of an i-motif sequence, dramatic changes in fluorescence occurred as the DNA structures changed from single-strands to i-motif quadruplex structures. Interestingly, the cis form of Azo-py induced a more stable i-motif structure than did the trans form, as confirmed from circular dichroism spectra and melting temperature data. The absorption and fluorescence signals of these Azo-py-incorporated i-motif systems exhibited switchable and highly correlated signaling patterns. Such isomeric structures based on Azo-py might find potential applications in biology, where control over stable i-motif quadruplex structures might be performed with switchable fluorescence signaling.     

Modulation of i-motif thermodynamic stability by the introduction of UNA (unlocked nucleic acid) monomers

The influence of acyclic RNA derivatives, UNA (unlocked nucleic acid) monomers, on i-DNA thermodynamic stability has been investigated. The 22 nt human telomeric fragment was chosen as the model sequence for stability studies. UNA monomers modulate i-motif stability in a position-depending manner. The largest destabilization is observed for position C14, while UNA placed in position A12 causes significant increase of i-DNA thermodynamic stability. CD curves of UNA-modified variants imply no structural changes relative to the native i-motif.     

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.     

Deoxygenated phosphorothioate inositol phosphate analogs: synthesis, phosphatase stability, and binding affinity

Inositol phosphates, such as 1D-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], are cellular second messengers with potential roles in cancer prevention and therapy. It typically is difficult to attribute specific pharmacological activity to a single inositol phosphate because they are rapidly metabolized by phosphatases and kinases. In this study, we have designed stable analogs of myo-inositol 4,5-bisphosphate [Ins(4,5)P(2)] and Ins(1,4,5)P(3) that retain the cyclohexane scaffold, but lack hydroxyl groups that might be phosphorylated and have phosphate groups replaced with phosphatase-resistant phosphorothioates. An Ins(1,4,5)P(3) analog, 1D-2,3-dideoxy-myo-inositol 1,4,5-trisphosphorothioate, was synthesized from (-)-quebrachitol, and an Ins(4,5)P(2) analog, 1D-1,2,3-trideoxy-myo-inositol 4,5-bisphosphorothioate, was prepared from cyclohexenol. The Ins(1,4,5)P(3) analog was recognized by Ins(1,4,5)P(3) receptor with a binding constant (K(d)) of 810 nM, compared with 54 nM for the native ligand Ins(1,4,5)P(3), and was resistant to dephosphorylation by alkaline phosphatase under conditions in which Ins(1,4,5)P(3) is extensively hydrolyzed. Analogs developed in this study are potential chemical probes for understanding mechanisms of inositol phosphate actions that may be elucidated by eliciting specific and prolonged activation of the Ins(1,4,5)P(3) receptor.     

Oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid

Degradation of a synthetic oligodeoxynucleoside phosphorothioate was studied in six systems used for antisense inhibition experiments. Oligodeoxynucleoside phosphorothioates were degraded very slowly at 37 degrees C in all of the systems studied. Measured half-lives of pentadecamers were 12 +/- 1 h in rabbit reticulocyte lysate, 7 +/- 1 h in HeLa cell postmitochondrial extract, 14 +/- 2 h in RPMI 1640 with 10% fetal bovine serum, 8 +/- 1 h in undiluted fetal bovine serum, 9 +/- 1 h in adult human serum, and 19 +/- 7 h in rat cerebrospinal fluid.