Novel methods for nucleotide length control in double-stranded polyinosinic-polycytidylic acid production using uneven length components.

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.

Nucleic acid binding and other biomedical properties of artificial oligolysines.

In the present study, we report the interaction of an artificial oligolysine (referred to as AOL) realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI) and its complex with polycytidylic acid (poly I:C), RNAs with well-known interferon-inducing ability, and double-stranded (ds) DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD) and ultraviolet (UV) studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine-protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL.

Metallization of single-stranded polyI by Zn(2+) ions in neutral solutions.

Metallization of single-stranded polyinosinic acid (polyI) by Zn(2+) ions at pH 7.0 was studied by differential UV spectroscopy at different temperatures. It was found that polyI is metallized at N7 and N1 atoms of hypoxanthine. The concentration dependence of the degree of binding of Zn(2+) ions to both N7 and N1 sites was obtained, and the corresponding binding constants were determined. Metallization of N1 occurs due to Zn(2+) substituting the imino protons and is effective not only at alkaline but also at neutral pH. This makes multistranded polyI-based systems more promising candidates for use in nanoelectronics than natural DNA sequences, metallization of which can be achieved only at alkaline pH.

Interaction of 9-O-N-aryl/arylalkyl amino carbonyl methyl berberine analogs with single stranded ribonucleotides.

Studies on the molecular aspects of alkaloid-RNA complexation are of prime importance for the development of rational RNA targeted drug design strategies. Towards this goal, the binding aspects of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to four single stranded ribonucleotides, poly(G), poly(I), poly(C) and poly(U), were studied for the first time employing multifaceted biophysical tools. Absorbance and fluorescence studies revealed that these analogs bound non-cooperatively to poly(G) and poly(I) with binding affinities remarkably higher than berberine. The binding of these analogs to poly(U) and poly(C) was weaker in comparison to poly(G) and poly(I) but were one order higher in comparison to berberine. Quantum efficiency values revealed that energy transfer occurred from the RNA bases to the analogs upon complexation. The binding was dominated by large positive entropic contributions and small but favorable enthalpic contributions. Salt dependent studies established that the binding was dominated by hydrophobic forces that contributed around 90% of the total standard molar Gibbs energy. The chain length of the substitution at the 9-position was found to be critical in modulating the binding affinities. These results provide new insights into the binding efficacy of these novel berberine analogs to single stranded RNA sequences.

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex.

Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5-7.5 in the presence of NaCl at higher concentration than 50mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.

Quadruplex structure of polyriboinosinic acid: dependence on alkali metal ion concentration, pH and temperature.

The vibrational infrared (IR) absorption and vibrational circular dichroism (VCD) spectral changes of polyinosinic acid (polyI) as a function of alkali metal ion concentration, temperature and pH have been investigated to establish how changes in spectral features relate to the structural modifications of polyI. A single positive VCD couplet associated with the carbonyl absorption band is considered to be the signature of quadruplex structure for polyI. The disruption of the quadruplex structure with temperature increase or pH increase at low alkali metal ion concentration is evidenced by the disappearance of this positive VCD couplet. The absence of any VCD signal upon quadruplex disruption indicates that the newly formed structure lacks helical chirality and is likely to be disordered. In the presence of 1 M NaCl or 0.1 M NaCl, the heat-induced quadruplex disruption is completely reversible. A mildly alkaline environment, in the presence of 0.1 M NaCl, is not sufficient to support the quadruplex structure of polyI. Trehalose-assisted polyI film at room temperature exhibits the same quadruplex spectral signature as that seen for solution at room temperature, but the quadruplex spectral signature in the film state remains at higher temperature, unlike in solution. This indicates that the quadruplex structure of polyI in the film state resists heat-induced disruptions.

[The nature of different influence of Cd2+ ions on the conformation of three-stranded polyU-polyA-polyU and polyI-polyA-polyI in aqueous solution].

The methods of UV (DUV) spectroscopy and thermal denaturation were used to study the effect of Cd2+ ions on the conformational equilibrium of three-stranded (A21, A2U) and single-stranded (poly U, poly A and poly I) polynucleotides in aqueous solutions containing 0.1 M Na+ (pH 7). An analysis of the form and intensities of DUV-spectra of poly A, poly I and A2I revealed the presence of two types of complexes: interaction with N7 of purines, resulting in the formation of macrochelates and binding to N1 of poly A and poly I. Cd2+ ions do not bind to heteroatoms of A2U nitrogen bases, and, therefore, the conformation of its structure remains unchanged up to a concentration of Cd2+ 0.01 M. A "critical" concentration (1.5x10(-4) M) of Cd2+ ions exists above which A2I transits cooperatively into a new helical conformation, which has a lower thermostability. It is supposed that, during the formation of metallized A2I, Cd2+ ions form bridges between the adenine and hypoxanthine of its homopolynucleotide circuits, being arranged inside the triple helix.

Crystal structure of an RNA quadruplex containing inosine tetrad: implications for the roles of NH2 group in purine tetrads.

Polyinosinic acid has been known to adopt the four-stranded helical structure but its basic unit, inosine tetrad (I tetrad), has not been determined at the atomic level. Here we report the crystal structure of an RNA quadruplex containing an I tetrad at 1.4 A resolution. The I tetrad has one cyclic hydrogen bond N1...O6 with the bond length of 2.7 A. A water bridge is observed in the minor groove side of the base tetrad. Even though it is sandwiched by guanine tetrads (G tetrads), the I tetrad is buckled towards the 3' side of the tetrad plane, which results from the different interaction strength with K ions on two sides of the tetrad plane. Comparison with both G tetrad and adenine tetrad indicates that lack of NH2 in the C2 position makes the I tetrad prone to buckle for interactions with ligands. Two U*(G-G-G-G) base pentads are observed at the junction of the 5' termini of two quadruplexes. The uridine residue in the base pentad is engaged in two hydrogen bonding interactions (N2(G)-H...O2(U) and O2'(G)-H...O4(U)) and a water-mediated interaction (N3(G) and N3(U)) with the G tetrad. We also discuss the roles of amino group in purine tetrads and the inter-quadruplex interactions in RNA molecules. These quadruplexes may interact with each other by stacking, groove binding and intercalation.

Na+ and Mg2+ ion effect on the stability of a poly I . poly A . poly I triple helix.

Differential UV spectroscopy was used to study the temperature dependence of the conformational equilibrium in aqueous poly I . poly A . poly I (A2I) solutions containing Na+ (0.1-2 M) and Mg2+ (10(-5)-0.005 M) ions. Over the whole range of the studied Na+ and Mg2+ concentrations, the heating-induced destruction of the triple A2I helix is actually the A2I --> A + I + I (3 --> 1) transition. The rise of the transition temperature with increasing Na+ and Mg2+ contents is well described by Manning's and the "ligand" theories, which makes it possible to estimate the linear charge density on the single-stranded poly I (xi = 1.9 +/- 0.1) and the Mg2+-A2I binding constant (K = 1,250 M(-1) for the zero degree of binding). An analytical expression has been obtained, which correlates the constants of Mg2+ binding to three- and single-stranded polynucleotides (K3 and K1, respectively) and the linear charge density on them. There are only minor distinctions between the K3 values for A2U and A2I because these polynucleotides have similar structures. The difference in the K1 values is also slight as single-stranded poly U, poly I, and poly A have similar conformations. Dependence of the conformational transition temperatures of two triple helices with changing Mg2+ concentration.

Phonon dispersion in polyinosinic acid.

A study of the normal modes of vibration and their dispersion in polyinosinic acid [poly (I)] along the helix axis based on Urey-Bradley force field is reported. It leads to a better interpretation of Raman and FTIR spectra. A comparison of dispersion curves of poly (I) with poly (G) has been made. Characteristic features of dispersion curves, such as regions of high density-of-states, repulsion and character mixing are discussed. Predictive value of heat capacity as a function of temperature is reported.

[Some X-ray diffraction parameters of liquid-crystalline dispersions of nucleic acid-chitosan complexes ]. / Nekotorye rentgenograficheskie parametry zhidkokristallicheskikh dispersii kompleksov NK-khitosan.

Liquid-crystalline dispersions of nucleic acid-chitosan complexes (NA-chitosan) possess optical and X-ray diffraction properties different from those of "classical" cholesterics. It is possible that positive charge distribution (distance between charges, chitosan conformation, etc.) in the polymeric chain of chitosan molecule combining with NA molecule is the factor that affects the spatial structure of resulting dispersions.

Prediction of an HLA-DR-binding peptide derived from Wilms' tumour 1 protein and demonstration of in vitro immunogenicity of WT1(124-138)-pulsed dendritic cells generated according to an optimised protocol.

The Wilms' tumour 1 (WT1) protein is over-expressed in several types of cancer including leukaemias and might therefore constitute a novel target for immunotherapy. Recently, human leucocyte antigen (HLA) class I-binding WT1 peptides have been identified and shown to stimulate CD8(+) T cells in vitro. For maximal CD8 cell efficacy, CD4(+) helper T cells responding to major histocompatibility complex (MHC) class II-binding epitopes are required. Here, we report that scanning the WT1 protein sequence using an evidence-based predictive computer algorithm (SYFPEITHI) yielded a peptide WT1(124-138) predicted to bind the HLA-DRB1*0401 molecule with high affinity. Moreover, synthetic WT1(124-138)-peptide-pulsed dendritic cells (DC), generated according to a protocol optimised in the present study, sensitised T cells in vitro to proliferate and secrete interferon-gamma (IFN-gamma) when rechallenged with specific peptide-pulsed DC, but not with peripheral blood mononuclear cells (PBMC). These results suggest that the WT1 protein may yield epitopes immunogenic to CD4 as well as CD8 T cells, and therefore constitute a novel potential target for specific immunotherapy.

Study of the interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)Pt(II) complex with poly(I), poly(C) and poly(I) x poly(C).

The interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)platinum(II) (Ptpyrr) with the polynucleotides poly(I), poly(C) and poly(I) x poly(C) acids was studied by circular dichroism, molecular fluorescence and (1)H NMR spectroscopies. Multivariate Curve Resolution, a factor analysis method, was applied for the analysis and interpretation of spectroscopic data obtained in mole ratio and kinetics studies. This procedure allows the determination of the number of different interaction complexes present during the experiments and the resolution of both concentration profiles and pure spectra for all of them. Two different interaction complexes were observed at the experimental conditions studied. The first one, at low Ptpyrr:polynucleotide ratio (r(Ptpyrr:poly)) values, corresponds to the interaction of Ptpyrr with hypoxanthine bases in the poly(I) moiety. This interaction leads to the destabilization and dissociation of the double-stranded conformation. The second complex was observed at higher r(Ptpyrr:poly) values and corresponds to the interaction of Ptpyrr to cytosine bases in poly(C) moiety. The formation of both complexes showed that the interaction of Ptpyrr with hypoxanthine bases occurred at the first stages of the reaction and with cytosine bases at longer reaction times. The results obtained show the utility of the Multivariate Curve Resolution approach for the analysis of data obtained by monitoring spectroscopically the interaction equilibria of platinum compounds with nucleic acids.

Structure-function relationship of three triple-helical nucleic acids.

The nucleic acid triplexes poly d(T) x poly d(A) x poly d(T), poly (U) x poly (A) x poly (U), and poly (I) x poly (A) x poly (I) display a sort of continuity between each other. However, their morphologies present their own individuality which, considering those of their parent duplexes, are quite unexpected. This comparison helps to understand triplex structure-function relationship. While helical parameters are functions of the sugar pucker, low values of WC and Hoogsteen base-pair propellers is commonplace for triplexes and the Hoogsteen base-pair geometry monitors the effects of the interstrand phosphates charge-charge repulsion.

[Interaction of divalent metal ions with four-stranded polyriboinosinic acid]. / Vzaimodeistvie ionov dvukhvalentnykh metallov s chetyrekhtsepochechnoi poliriboinozinovoi kislotoi.

Interaction of Mg2+, Ca2+, Cu2+ ions with the four-stranded poly(I) was studied using differential UV and visible spectroscopies. It was shown that, up to concentrations of approximately 0.1 M, Mg2+ and Ca2+ ions do not bind to heteroatoms of hypoxanthine of the four-stranded poly(I). Cu2+ ions interact with N7 (and/or N1) and O6 (through the water molecule of the hydrate shell of the ion). The latter seems to induce the enolization of hypoxanthine the deprotonation of N1, and, as a result, the transition of the four-stranded helix to single-stranded coils. Single-stranded chains form compact particles with an effective radius of about 100 A.

Structure of poly (I).poly (A).poly (I).

The molecular structure of poly (I).poly (A).poly (I) has been determined and refined using the continuous intensity data on layer lines in the x-ray diffraction pattern obtained from an oriented fiber of this polymorphic RNA complex. The polymer forms a 12-fold right-handed triple-helix of pitch 39.7A and each base-triplet is stabilized by quasi Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in all the three strands have C3'-endo conformations. The final R-value for this best structure is 0.24 and the x-ray fit is significantly superior to all the alternative structures where the different chains might have different furanose conformations. This all-purine triple-helix, counter-intuitively, has a diameter roughly 3A shorter than that of DNA and RNA triple-helices containing a homopurine and two complementary homopyrimidine strands. Its compact, grooveless cylindrical shape is consistent with the lack of lateral organization.

PMTI, a broadly active unusual single-stranded polyribonucleotide, inhibits human immunodeficiency virus replication by multiple mechanisms.

Poly(1-methyl-6-thioinosinic acid), or PMTI, is a single-stranded polyribonucleotide and is the first homopolyribonucleotide devoid of Watson-Crick hydrogen bonding sites to show potent human immunodeficiency virus (HIV) inhibition. PMTI was found to be active when evaluated against a variety of low passage clinical HIV isolates in fresh human peripheral blood cells, including T cell-tropic and monocyte-macrophage-tropic viruses, syncytium-inducing and non-syncytium-inducing viruses and viruses representative of the various HIV-1 clades (A through F). The compound was active against HIV-2, all nucleoside and non-nucleoside reverse transcriptase (RT) inhibitor drug-resistant virus isolates tested and interacted with AZT or ddl to synergistically inhibit HIV infection. In biochemical inhibition assays, PMTI was determined to be a potent inhibitor of HIV-1 and HIV-2 RT, including RTs with mutations that engender resistance to nucleoside and non-nucleoside RT inhibitors. PMTI inhibited both the polymerase and RNase H activities of HIV RT. PMTI did not inhibit HIV-1 protease or integrase. Cell-based mechanism of action assays indicated that PMTI also interfered with early events in the entry of HIV into target cells. Furthermore, PMTI inhibited the fusion of gp120-expressing and CD4-expressing cells, but at concentrations approximately 1 log10 greater than those that inhibited virus entry. These results suggest that the homopolyribonucleotide PMTI blocks HIV replication in human cells at its earliest stages by multiple mechanisms, inhibition of virus entry and inhibition of RT.

Binding studies of a triple-helical peptide model of macrophage scavenger receptor to tetraplex nucleic acids.

The macrophage scavenger receptor (MSR), involved in the uptake of oxidized LDL, binds a variety of polyanionic ligands, and in particular shows selectivity for tetraplex forms of nucleic acids. The ligand binding region has been shown to lie in the triple-helical collagen-like domain of MSR. A model peptide-nucleic acid system is presented here to clarify how the triple-helical motif of MSR recognizes and binds tetraplex nucleic acids. The triple-helical peptide MSR-1, with the sequence (POG)3PKGQKGEKG(POG)4, contains a nine amino acid basic sequence implicated in MSR ligand binding, flanked by Pro-Hyp-Gly triplets to provide stability. The ability of this triple-helical MSR-1 peptide to bind to and perturb the conformation of nucleic acids in tetraplex, duplex, and single-stranded states was assessed by monitoring changes in the nucleic acid circular dichroism spectrum in the 240-300 nm region. Our results show that the triple-helical MSR-1 peptide binds to tetraplex poly(I) in a stoichiometric manner and is capable of reproducing the discrimination exhibited by the native MSR molecule for tetraplex over double-stranded or single-stranded nucleic acid states. The triple-helical reference peptide (POG)10 does not bind to tetraplex poly(I), suggesting that binding requires the highly basic 9-mer sequence from MSR that is included in MSR-1. The MSR-1 peptide did not perturb the CD spectra of a series of other tetraplex nucleic acids, indicating that it does not model the broader specificity that MSR shows under physiological conditions. Models of possible interactions between a triple-helical peptide and a tetraplex polynucleotide are proposed on the basis of the stoichiometry observed for the complex between triple-helical MSR-1 and tetraplex poly(I).

A resonance Raman spectroscopic study of the quadruplex form of polyriboinosinic acid.

The four stranded form of polyriboinosinic acid, or poly(rl), formed under conditions of high ionic strength, has been studied principally by resonance Raman spectroscopy excited in the ultraviolet absorbent band of the hypoxanthine residues. UV Absorption and circular dichroism studies were made, principally in order to verify the presence of the quadruplex form at the low concentrations of poly(rl) used, and a trial experiment with the structural probe Tb3+ was also performed. Experimental evidence is found for highly stacked metastable forms present at low concentrations of polynucleotide, which are destroyed by heating in favor of the two well known forms.

A vibrational spectroscopic study of the metastable form of associated polyinosinic acid.

We have studied by Raman and ir spectroscopy the metastable complex formed by the self-association of polyinosinic acid in aqueous solution. The complex is easily prepared by quickly cooling to ca. 0 degrees C a warm solution of the polyribonucleotide to which a small amount of rubidium salt has been added. Upon heating, this metastable form melts cooperatively near 13 degrees C, well below the dissociation temperature of a stable four-stranded complex, which occurs at 47 degrees C in the same conditions. The presence of several components in the stretching-mode region of the carbonyl groups in the vibrational spectra of the metastable complex suggests that it also has a parallel four-stranded structure. The difference in structure between the two forms is believed to be caused by the presence of fewer metal ions in the central channel of the metastable complex, in agreement with conclusions reached in previous investigations. The Raman spectra further show that the ribose units in the metastable form have a C3'-endo conformation, in contrast with the stable form, for which we have previously suggested a mixed C2'-endo/C3'-endo conformation.