A circular dichroism study of poly dG, poly dC, and poly dG:dC

We have measured the ultraviolet circular dichroism spectra of oligo d(pG)₅, poly dN AcG, poly dI, poly dC, two samples of poly dG, and four samples containing double-stranded poly dG:dC. We find that oligo d(pG)₅ and poly dG exist in self-complexed forms as well as in single-stranded forms. Unlike the self-complexed form of poly dG, the single-stranded form of poly dG can hydrogen-bond with single-stranded poly dC. We present spectral data for double-stranded poly dG:dC, which can be used to help characterize poly dG:dC preparations and which provide a basis for resolving discrepancies among other reported poly dG:dC spectra.     

Comparison of oligoribonucleotides and oligodesoxyribonucleotides. I. Formation of double helices

The ability of oligodesoxyribonucleotides of various chain lengths to form complexes has been compared with that of oligoribonucleotides. Four series of oligonucleotidcs were prepared and investigated, i.e., dC_n at acid pH versus rC_n, dA_n and dT_n versus. rA_n and rU_n at neutral pH. The results indicate that in dilute solution, the formation of complexes is greatly facilitated in the case of desoxyoligomers and occurs for shorter oligomere than in the corresponding ribooligomers. The spectrophotometric titration of deoxyribooligo C indicates the appearance of two pK values in the 4–5 pH region characteristic of the double-stranded form, which occurs for much shorter dC_n than rC_n. The circular dichroism (CD.) spectra of deoxycytidylies in dilute solution starting from the trimer are conservative, characteristic of the double-stranded helical form of poly C at acid pH. In contrast, the CD spectra of a series of corresponding ribo C_n, under identical conditions is of nonconservative character similar to that of the single-stranded form of poly C at neutral pH, but differs in the band position. This spectrum is called intermediate. Only at higher concentrations of oligonucleotidcs (i.e., 10^?3Minstead of 10^?4M) does the circular dichroism spectrum of longer ribocytidylics assume conservative character. Thermal denaturation of deoxycytidylces at acid pH are strongly dependent on chain length and concentration, its one would expect for a cooperative helix-coil transition. The circular dichroism spectra measured at different temperatures shows one isosbestic point. In dilute solution, the standard-state enthalpy change found was 5–6 kcal/mole for higher oligomers (dC₇). These properties are all in agreement with a structural transition from the d-C_n double-stranded form to a coil for n > 3. Studies of dA_n and dT_n in solutions of high ionic strength at low temperature indicate that complex formation occurs already at the level of trimer and for high oligomers. Under identical conditions a complex between rA_n and rU_n is detected only for oligomers longer than the hexamer. The nature of the “intermediate” form of oligoribo C at acid pH and low temperature was investigated by sedimentation and circular dichroism. A model of rC_n is proposed of linear molecules which are partially double-stranded and partially single-stranded, which probably are slowly rearranged by “slippage” into a regular-double-stranded helical form.     

Stability and Transformations of bis-PNA/DNA Triplex Structural Isomers

Abstract

Structurally isomeric complexes formed between homopyrimidine bis-PNAs (T₂JT₂JT₄-linker-T₄CT₂CT₂) and single- and double-stranded DNA targets were investigated. These complexes are triplexes designated S1, S2 and S3 in order of increased mobility by polyacrylamide gel electrophoresis. It is shown that the S3 isomer is formed only on double-stranded DNA and possesses highest stability. Isomers S2 and S1 are formed upon binding of bis-PNA to double-stranded as well as to single-stranded DNA. It was found that the stability of the isomer S1 increases dramatically in the presence of excess single-stranded oligonucleotide complementary to the bis-PNA. The structure of the stabilized S1 isomer is proposed to consist of two bis-PNA/DNA triplexes. The relationship between the yield of the isomer S1 formed on single-stranded DNA and the bis-PNA concentration was investigated and a kinetic model of the formation of S1 is presented.     

Studies of the Activity of Peroxidase‐Like DNAzyme by Modifying 3′‐ or 5′‐End of Aptamers

In the presence of hemin and under appropriate conditions, some modalities of G‐quadruplexes can form a peroxidase‐like DNAzyme that has been widely used in biology. Structure? function studies on the DNAzyme revealed that its catalytic ability may be dependent on the unimolecular parallel G‐quadruplex. In this report, we present the preliminary investigation on the relationship between the structure and function of DNAzymes through a terminal oligo modification in G‐quadruplex sequences by adding different lengths of oligo‐dT to the 3′‐ or 5′‐end of the aptamers. The results suggested that adding dT_n to the 5′‐end of the DNA sequence of the enzyme improved the ability of hemin to bind with DNA, but the addition of dT_n to the 3′‐end decreased the binding ability of hemin for DNA. The increased stability of the assembled DNAzyme would lead to more favorable binding between the enzyme and substrate (H₂O₂), facilitating higher peroxidase activity; on the contrary, with lower stability of the DNAzyme complex, we observed reduced peroxidase activity.     

Optimization of subtractive hybridization in construction of subtractive cDNA libraries

The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C_ot values.     

Denaturation of RNA with dimethyl sulfoxide

The denaturation of single-stranded and double-stranded RNA's in solutions with varying proportions of dimethyl sulfoxide has been followed by changes in absorbancy, optical rotation, and—with a double-stranded form of bacteriophage of MS2 RNA— infectivity for bacterial spheroplasts. By these criteria the RNA's studied, including the synthetic polynucleotide rG:rC, are completely denatured at room temperature in high concentrations of this solvent. In lower concentrations, the T_m of the RNA preparation is decreased only slightly as the dimethyl sulfoxide concentration is raised until a critical concentration is reached. The T_m falls sharply with small further increases in dimethyl sulfoxide concentration. Sedimentation studies can be conducted directly in these media. The determination of sedimentation velocity in 99% dimethyl sulfoxide containing 0.001M EDTA provides a reliable estimate of RNA molecular weights.     

Specificity of DNA-basic polypeptide interactions. III. Sequential and random copolymers of lysine and aromatic amino acids.

Sequential and random lysine copolymers containing various amounts of different aromatic amino acids were synthesized. The sequential copolypeptides exhibited strong dependence of yield and degree of polymerization on the amino acid sequence of the repeating unit. To elucidate the specific contributions of aromatic side chains to the interaction of these copolymers with DNA, direct-mixed complexes were studied by thermal denaturation and CD. The melting behaviour of peptide-bound DNA was found to be strongly affected by amino acid composition and sequence. The contribution of the different aromatic amino acids to thermal stability decreased in the order: polylysine > [Lys, Tyr]_n > [Lys,Phe]_n > [Lys,(OMe)Tyr]_n. The CD spectrum of DNA was altered by random copolymers, whereas sequential copolymers exhibited no changes. The influence of the random copolymers on the CD spectrum of DNA decreased in the series: polylysine > [Lys,Phe]_n > [Lys,(OMe)Tyr]_n > [Lys,Tyr]_n. The contribution of the different aromatic amino acids to thermal stability is interpreted as stacking tendencies toward denatured and, in the case of Tyr, H-bond formation with native DNA. The differences found for the random and the sequential polypeptides can best be explained by assuming a cooperative action of rather small peptide segments.     

Solubilities of amino acids

The Mettler/Paar precision density meter DMA-02D has been used to determine the concentration of saturated solutions of amino acids at 20.0, 25.0, and 29.8 °C. The technique has proven itself an elegant and precise method. The solubilities of all of the amino acids with the exceptions of proline, lysine, and cystine have been measured. The Gibbs free energies of transfer from saturated water solution to 1M Na₂SO₄ and to 1M Gu·HCL along with the van't Hoff heats and entropies have been calculated. The van't Hoff heats have been compared with the calorimetrically determined heats for some of the amino acids. The Lumry-Rajender relation between the entropy and heats has been observed. The process of transfer of the amino acids from water to the solvents is primarily enthalpic rather than entropic.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Characterization of the HeLa cell single-stranded DNA-dependent ATPase/DNA helicase II

A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K. and Baril, E.F. (1990) Biochem 29, 8753–8759). Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Caoet al. (1994) Biochem 33, 8548–8557) and identity of the Ku protein as the human DNA helicase II (Tutejaet al. (1994) EMBO J. 13, 4991–5001) have been reported recently. Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II. Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits. The p90 subunit is predominantly nuclear and is easily dissociated from chromatin. The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix. Both the p90 and p70 subunits of the ATPase are present in G₁ and S phase of the cell cycle and are rapidly degraded in the G₂/M phase of the cell cycle.Abbreviations ssDNA single-stranded DNA - dsDNA double-stranded DNA - ATPase adenosine triphosphatase - HDH II human DNA helicase II - PGK 3-phosphoglycerate kinase     

Statistical mechanical deconvolution of thermal transitions in macromolecules. III. Application to double-stranded to single-stranded transitions of nucleic acids

The statistical mechanical deconvolution theory for macromolecular conformational transitions is extended to the case of nucleic acids transitions involving strand separation. It is demonstrated that the partition function, Q, as well as all the relevant thermodynamic quantities of the system, can be calculated from experimental scanning calorimetric data. In particular, it is shown that important thermodynamic parameters such as the size of the average cooperative unit during strand separation, the mean helical segment length, and the mean coil-segment length can be calculated from the average excess enthalpy function 〈ΔH〉. The theory is applied to the double-stranded to single-stranded transition of the system poly(A)·poly(U) at different salt concentrations. It is shown that the mean helical segment length is a monotonically decreasing function of the temperature well before strand separation occurs. On the other hand, the mean coil segment length remains practically constant until temperatures very close to T_m. Both experimental findings clearly indicate that the unfolding of poly(A)·poly(U) proceeds through the formation of many short helical sequences. The cooperative unit for the strand separation is calculated to be about 120 base pairs and essentially independent of the salt concentration. The existence of a minimum helical segment length of 10 ± 2 base pairs within the double-stranded form is calculated.     

The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice

Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb α_S1-casein/human lysozyme DNA construct and a small 5.5 kb β-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.     

Double-stranded RNA

Summary High molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplomaviruses); virus-specific dsRNA is also found in cells infected with single-stranded RNA viruses.DsRNA has been identified in a variety of apparently normal eucaryotic cells and is associated with the killer character of certain strains of Saccaromyces cerevisiae.The properties and significance of these various dsRNA species are described and discussed, as well as the available information concerning the biosynthesis of such RNA in virus-infected cells, its degradation by a variety of enzymes, and some problems concerning the variables which may control this process.Finally, the biological functions of dsRNA are briefly considered, as well as the structural properties important for its activity as an inducer of interferon and an inhibitor of protein synthesis.Abbreviations dsRNA for double-stranded RNA - ssRNA for single-stranded RNA - SSC for 0.15 m sodium chloride, 0.015 m sodium citrate, pH 7 - Poly(A), poly(C), poly(U) for polyadenylate, polycytidylate and polyuridylate, respectively - Poly(A).poly(U), poly(G).poly(C), poly(I).poly(C) for double-stranded complexes formed between polyadenylate and polyuridylate, polyguanylate and polycytidylate, and polyinosinate and polycytidylate, respectively. - Poly(rA).poly(dT) for the complex formed between polyriboadenylate and polydeoxyribothymidylate - Poly(A-U), poly(G-C) for the alternating copolymers containing AMP and UMP, or GMP and CMP, respectively - Poly(rA).poly(dUz) for the complex formed between polyadenylate and poly 2-azido-2deoxyuridylate - (I)_n.(br⁵C)_n for the complex formed between polyinosinate and poly 5-bromocytidylate - (I)_n.(s²C)_n for the complex formed between polyinosinate and poly 2-thiocytidylate - (dI_n3)_n.(C)_n for the complex formed between poly 2-azido-2-deoxyinosinate and polycytidylate - MW for molecular weight     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Infrared spectra of isotopic polyglycines

For infrared absorption measurements, the following five isotopic polyglycines have been prepared: ordinary polyglycine (—NHCH₂CO—)_n, N-deuterated polyglycine (—NDCH₂CO—)_n, C-deuterated polyglycine (—NHCD₂CO—)_n, completely deuterated polyglycine (—NDCD₂CO—)_n, and N¹⁵-substituted polyglycine (—¹⁵NHCH₂CO—)_n. Infrared spectra have been observed both in the I and II forms of each of these five isotopic polyglycines in the spectral region of 4000–300 cm.^?1. On the basis of the comparison of these spectra with each other, a nearly complete set of assignments of the observed bands of polyglycines has been given.     

Characteristics of immobilized invertase

Five kinds of immobilized invertases (IMI)—covalently of porous glass and ion-exchange resins and ionically on ion-exchange resins—have been prepared and their kinetic characteristics for sucrose hydrolysis, such as K_m, K, pH profile, and thermal stability were studied. Comparing the values of K_m and activation energy and the entropy of IMI with those of native invertase, it was concluded that the immobilization influences not binding but kinetic specificity. The effects of the immobilization method on thermal stability were also discussed.     

Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

The effect of physiological concentrations of KCl and MgCl₂ on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl₂ concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl₂ indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation. Received: October 12, 1997 / Accepted: January 29, 1998     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

Binding of Nucleic Acids to Intermediate Filaments of the Vimentin Type and Their Effects on Filament Formation and Stability

Abstract

Guanine-rich polynucleotides such as poly(dG), oligo(dG)_12–18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)₂₅ was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG)·poly(dC), and, of those of the RNA type, preferentially poly(rA)·poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.     

CD studies on the conformation of some deoxyoligonucleotides containing adenine and thymine residues

CD studies of the deoxyoligomer series d(pT)_n and d(pA)_n show increasing CD maxima for oligo (dT)'s with chain length variation from two to seven, while oligo (dA)'s exhibit a decreasing CD maximum. Concentrated solutions of NaClO₄ cause a decrease in the CD of longer oligo (dT)'s towards the CD of d(pT)₂ which is different from oligo dA's. Probably base-sugar interactions are important in the observed conformational effects. The chemically synthesized oligomers dpApApTpT and dpTpApTpA show deviations in their CD spectra which reflect a dominating conformational effect of d(pA)₂ in the former but not in the alternating isomer.