Paul O. P. Ts'o

Physicochemical basis of the recognition process in nucleic acid interactions: I. Interactions of polyuridylic acid and nucleosides

Interaction of adenosine and poly U† was studied by equilibrium dialysis, solubility, optical rotation, ultracentrifugation and viscosity measurements. Only when a threshold concentration of adenosine is reached does binding of adenosine to poly U take place in a co-operative manner, with a stacking energy of 5 to 6 kcal. estimated from the adsorption isotherm. The complex thus formed has an ordered structure, presumably helical, and, as expected, a molecular weight higher than that of poly U. The stability of the complex, besides being highly dependent on the concentration of adenosine, is influenced by ionic strength and temperature in a manner similar to the system of polynucleotide interaction. The stoichiometry of the complex is 1A to 2U at 5°C and becomes 1A to 1U at 20°C. This interaction is specific, with the possible involvement of N-1,6-amino and N-7 of adenosine in the hydrogen-bonding scheme, while the pentose moiety has a relatively insignificant role. Other studies from our laboratories have shown that both purines and nucleosides associate to form stacks in aqueous solution as a result of hydrophobic and stacking energy. It is proposed that these adenosine stacks are bound to poly U through specific hydrogen bonding. Thus, both hydrogen bonding and hydrophobic stacking energy co-operate and complement each other in providing the driving forces for this interaction.

Interaction of poly-l-lysine and nucleic acids

Interactions of poly- L -lysine with poly (I + C) and with thymus DNA in dilute solutions and in low ionic strength have been studied. The helix-coil transition of the polynucleotides in the reaction was followed by optical density at 248 mμ, or 260 mμ Both of these poly- L -lysine plus polynucleotide solutions gave a two-step transition, one of which (at a lower temperature) corresponds to the melting of the free polynucleotide double helix and the other (at a higher temperature) corresponds to the melting of a poly- L -lysine-polynucleotide complex. These transition temperatures are independent of the poly- L -lysine concentration added. However, as the poly- L -lysine concentration increases in the mixture, the hyperchromicity at the first transition decreases proportionately, together with a corresponding increase of hyperchromicity at the second transition. The poly- L -lysine-polynucleotide complex and the free polynucleotide were resolved by means of sucrose density-gradient electrophoresis. The behavior of the DNA molecules in the binding reaction can be monitored by the help of 32P-labeled DNA in the electrophoresis experiments. These results show that at room temperature and in a dilute salt solution, the poly- L -lysine-polynucleotide binding reaction is quantitative, irreversible and with a definite stoichiometric ratio. The lysine/nucleotide ratio in the poly- L -lysine-DNA complex was determined to be 1 : 1 and in the poly- L -lysine-poly (I + C) complex to be 0-5 : 1.

Interaction of nucleic acids: VIII. Binding of magnesium ions by nucleic acids☆☆☆

Abstract The lowering of the activity of magnesium ions in aqueous solution due to the presence of nucleic acids (5 to 20 m m ) was measured by a divalent cation specific electrode which shows a large decrease in potential. The electrode response over a wide range of MgCl2 concentration (10−1 to 10−4 m ) in water is linear as described by the Nernst equation. The binding studies were carried out in ~ 5 m m -sodium phosphate buffer, at neutral pH, with a variety of nucleic acids. The results are treated as site-binding to the phosphate in a multiple equilibrium using the equation of Scatchard (1949). The binding isotherms in the Scatchard plot (v/A0 versus v, where v is the degree of saturation of binding sites and A0 is the concentration of free Mg2+) show a limited linear portion from approximately 30 to 70% saturation of binding sites and then the slopes decrease markedly. As a first-order approximation to account for the interference factor (electrostatic, conformational, etc.) in the binding process of magnesium ions by the polymer with only one type of binding site (the phosphate ligand), two additional treatments were added to the Scatchard equation: (1) the binding constant, k, is replaced by ke−φv where φ represents the interference factor; (2) the derivative of the equation was taken to construct a [(δ v /A 0 )/δ v ] n versus v plot. In these plots, there is a region of zero slope (horizontal region) and a region of negative slope(s). The value from the horizontal region is taken as the apparent intrinsic constant for magnesium ion binding by the nucleic acids in the neutral sodium phosphate buffer at room temperature: transfer RNA (≈1.7 × 104) > poly A ≈ poly A· poly U ≈ poly I ≈ poly I · poly C ≈ native DNA ≈ stabilized stored denatured DNA (≈6 × 103) > freshly denatured DNA ≈ poly U ≈ poly C ≈ (≈3 × 103) ⪢ monomers (≈2 × 102). The slope in the derivative plot is related to the quantity −φ, varying from −φ to −2φ. The slopes for all the nucleic acids are similar except for transfer RNA, which has a much larger slope. These results indicate the importance of the conformation of nucleic acid in the binding of magnesium ions as reflected in both the apparent binding constant and the interference factor. Since the binding constant and the interference factor of transfer RNA are larger than those of other nucleic acids, these observations indicate that transfer RNA has a more folded conformation.

Structural requirements of the rIn·rCn complex for Induction of human interferon☆

Abstract Two requirements for the polyriboinosinate and polycytidylate complex (rIn·rCn) to induce human interferon in cell cultures are generally assumed: (1) intactness of the double-stranded complex (with a Tm higher than the incubation temperature); (2) adequate resistance to nucleases. In this study, the rIn or rCn strand in the complex was interrupted either by unpaired bases (U or G) or by bond breakage. These imperfect complexes have Tm values substantially higher than 37 °C and can be protected from nucleases by complex formation with polylysine; this complex formation does not impair the induction ability of rIn·rCn. The r(I39,U)n·rCn and r(I21,U)n·rCn complexes were found to have little induction ability, while rIn·r(C20,G)n, rIn·r(C22,U)n, rIn·(C13,U)n and rIn·r(C7,U)n were found to be active. Similarly, rIn·oligocytidylate complexes (e.g. rIn·(Cp)23G > p) were active, while oligoinosinate rCn complexes (e.g. rI19·rCn) generally were not. Addition of poly l - (or d -) lysine to these imperfect complexes, although rendering them resistant to enzymic destruction, did not increase their antiviral activity. Thus, for interferon induction, the structural requirements in strand continuity and base-pairing are apparently more stringent in the rIn strand than in the rCn strand. Two inducers, rIn·r(C13,U)n and rIn·r(C20,G)n, may have a better therapeutic index than rIn·rCn itself; they are hydrolyzed by nucleases five- to eight-fold faster, and yet are nearly as active as interferon inducers.

A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation.

Abstract A mammalian cellular system, utilizing Syrian hamster embryo cells, was developed for the concomitant study of neoplastic transformation and somatic mutation. Chemically induced somatic mutation of the cells was assayed at two genetic loci. Mutants deficient in hypoxanthine phosphoribosyl transferase (HPRT) were detected by the production of colonies resistant to 8-azaguanine (AGr) or 6-thioguanine (TGr) and mutants with an altered Na+/K+ ATPase were detected by the production of colonies resistant to ouabain (Ouar). Colonies resistant to each of the three selective agents were isolated and characterized. AGr and TGr resistant cells maintained their resistance to the selective agent after isolation and growth in the absence of the drug, displayed a low reversion frequency, and possessed less than 1% of the HPRT activity of the wild-type cells. AGr cells were also resistant to the cytotoxicity effects of 6TG. Ouar cells also maintained their resistance to ouabain and were less sensitive to the inhibition of 86Rb uptake by ouabain than the wild-type cells. The spontaneous frequency of all three types of resistant cells was The factors involved in the quantification of the mutation frequencies of hamster embryo cells following exposure to carcinogens were determined. Cytotoxicity was assayed by a reduction in the cloning efficiency of the treated cells. The recovery efficiencies of the resistant cells were measured by reconstitution experiments and the degree of cross feeding effects of HPRT− cells was determined. The expression time of the mutations following exposure of the cells to carcinogens was also examined, and the mutation frequencie at the two loci of hamster embryo cells following exposure to MNNG or benzo(a)-pyrene (B(a)P) were determined. Employing this system, a quantitative comparison can be made between the frequencies of somatic mutation and morphological transformation.

In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity

Normal diploid Syrian hamster dermal mesenchymal cell strains, regardless of the age of the tissue of origin, exhibit in vitro cellular senescence. The frequency of spontaneous escape from senescence and conversion to a permanent cell line is less than 5% among replicate flasks. The overall pattern of senescence of cells of fetal, neonatal, young adult (6 months) and aged adult (24 months) origin is similar in terms of the morphological changes and proliferative changes indicated by the reduction of saturation density, cloning efficiency and [3H]thymidine labeling index and by the increase in population doubling time and cell volume. However, the average maximum cumulative population doubling level is characteristic for each cell type: 13-day gestation fetal cells, 28.6; neonatal cells, 18.7; young adult cells, 13.8; aged adult cells, 11.1. Thus, the in vitro proliferative capacity of Syrian hamster mesenchymal cells is inversely related to the in vivo age of the donor.

The effects of interferon on epidermal growth factor action

Abstract Epidermal growth factor-stimulated thymidine incorporation in human fibroblasts is inhibited more than 80% by human interferon, whereas the stimulation of α-aminoisobutyrate uptake is unaffected. Maximum inhibition of thymidine incorporation is observed after treatment of cells with interferon prior to the onset of DNA synthesis. However, even after the initiation of DNA synthesis, interferon rapidly blocks any further increase in thymidine incorporation. Despite these effects, interferon treatment causes no alterations in epidermal growth factor binding, receptor downregulation or receptor reappearance.

Chapter 30. Oligonucleotide Inhibitors of Gene Expression in Living Cells: New Opportunities in Drug Design

Publisher Summary This chapter describes some of the oligomers and experiments in which they have been used to control gene expression at the messenger RNA (mRNA) level, particularly in living cells. It appears that this concept could be exploited to develop clinically useful drugs to control the growth of viruses or malignant cells. Present day drugs that bind or intercalate in the minor groove of DNA have their limited ability to recognize specific nucleic sequences. In that way, they cannot take full advantage of the sequence information available in DNA, and thus are unable to inhibit expression of specific genes. The preparation, introduction, and expression of anti-sense RNAs require rather sophisticated molecular biology techniques. They can be prepared in the laboratory and then introduced into cells by microinjection or expressed in cells after transfection with plasmids carrying an anti-sense gene. The control of gene expression can, in theory, be achieved by single-stranded nucleic acids, that can specifically read complementary mRNA sequences via Watson–Crick base-pairing interactions. There exists a simpler approach that involves the use of short anti-sense oligonucleotides or oligonucleotide analogs prepared by chemical synthesis. There are three types of oligonucleotides that are oligodeoxyribonucleotides, oligomers having modified sugar–phosphate backbones, and oligomers derivatized with specific functional groups. Little is known about the stability, distribution, acute and/or chronic toxicity, or immunogenicity of compounds in vivo belonging to the anti-sense oligonucleotides or oligonucleotide analogs class. The promising results obtained in cell culture studies suggest that anti-sense oligonucleotide analogs offer a unique approach for the rational design of drug.

DNAase I encapsulated in liposomes can induce neoplastic transformation of syrian hamster embryo cells in culture

We have used liposomes to deliver DNAase I inside normal Syrian hamster embryo (SHE) cells. We showed the entrance of DNAase I inside the cell by dose-dependent cytotoxicity; and the entrance of DNAase I into the nucleus by the induction of chromosomal aberrations and somatic mutation at the HPRT locus (but not at the Na+/K+ ATPase locus). The induction of neoplastic transformation in cultures treated by DNAase I-in-liposomes was manifested by increased saturation density, colony formation at low seeding density, colony formation in 1% serum and 0.3% agar, and tumorigenicity in 100% of injected animals. The acquisition of anchorage-independent growth became apparent only after 39-57 posttreatment population doublings. Thus damage to DNA alone can initiate the neoplastic transformation process; but for full expression of the neoplastic phenotypes, a long progression time is required for the acquisition of anchorage-independent growth and tumorigenicity.

Proton NMR and Optical Spectroscopic Studies on the DNA Triplex Formed by d-A-(G-A)7-G and d-C-(T-C)7-T

Triplex and duplex formation of two deoxyribohexadecamers d-A-(G-A)-G (a) and d-C-(T-C)-T (b) have been studied by UV, CD, fluorescence, and proton NMR spectroscopy. Optical studies of a and b at dilute concentrations (microM range) yielded results similar to those seen for polymers of the same sequence, indicating that these hexadecamers have properties similar to the polymers in regard to triplex formation. The CD spectra of concentrated NMR samples (mM range) are similar to those observed at optical concentrations at both low and high pH, making possible a correlation between CD and NMR studies. In NMR spectra, two imido NH-N hydrogen bonded resonance envelopes at 12.6 and 13.7 ppm indicate that only the duplex conformation is present at pH greater than 7.7. Four new NH-N hydrogen-bonded resonance envelopes at 12.7, 13.5, 14.2, and 14.9 ppm are observed under acidic conditions (pH 5.6) and the two original NH-N resonances gradually disappear as the pH is lowered. Assignment of these four peaks to Watson-Crick G.C. Hoogsteen T.A Watson-Crick A.T, and Hoogsteen C+.G hydrogen-bonded imidos, respectively, confirm the formation of triple-stranded DNA NMR results also show that triplex is more stable than duplex at the same salt condition and that triplex melts to single strands directly without going through a duplex intermediate. However, in the melting studies, a structural change within the triple-stranded complex is evident at temperatures significantly below the major helix-to-coil transition. These studies demonstrate the feasibility of using NMR spectroscopy and oligonucleotide model compounds a and b for the study of DNA triplex formation.