Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads.

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.

Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion.

Monoliths are considered as a novel generation of stationary phases. They were applied for capillary electrochromatography and liquid chromatography exploiting every action principle such as ion-exchange, affinity recognition, reversed-phase, and hydrophobic interaction. The fast separation was explained by convective transport of the solutes through the bed. The contribution of this mode of transport is similarly explained as done for the beds packed with particles with gigapores. For monolithic beds, the concept of an ultrashort bed was frequently used. This mode of operation allows very short separation time. In many cases a gradient elution is necessary to achieve separation. Examples of applications for protein and polynucleotide separation performed on monoliths are given. Enzymatic conversion was described showing the examples of several immobilzed enzymes.

Properties of a polynucleotide synthesized by strain 74A of Neurospora crassa.

A polynucleotide (or a fragment of RNA) was purified to apparent homogeneity by HPLC from mycelium of the wild strain 74A of the mould Neurospora crassa, after growth on sucrose and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 hr at 30 degrees. The M(r) was ca 20,000 as determined by HPLC at pH 6.8. Polynucleotide synthesis ranged from 4.0 to 6.5 micrograms polynucleotide per mg dry mycelium in mycelium of the wild strain 74A and the various phosphorus regulatory and structural mutant strains of the mould N. crassa. Kinetic data showed that the polynucleotide interacts with mycelial Pi-repressible alkaline phosphatase by inhibiting its p-nitrophenylphosphatase activity and by protecting the enzyme against thermal inactivation in the presence of high concentrations of ammonium sulphate.

High performance liquid chromatography of nucleic acids and the related compounds on a fluorinated silica gel column.

Separations of nucleic acids and the related compounds were investigated by HPLC on a new fluorinated bonded silica gel column. Polyadenylate (Poly (A)) enzymatic partial hydrolysate sample and the mixture of various polynucleotide samples were sufficiently separated by the reversed-phase mode using a gradient elution with aqueous ammonium acetate/acetonitrile system. Mixed-mode separation on the fluorinated bonded phase coated with a tert-alkylammonium salt was also examined for the separation of the various polynucleotides including tRNAs.

Polymer support synthesis. XII. Derivatization with the 4-decyloxytrityl group as an aid in the affinity chromatography of oligo- and polynucleotides.

In a continuation of previous studies on trityl groups substituted with long alkyl chains as affinity-protecting groups for oligonucleotides, the use of the (4-decyloxyphenyl)diphenylmethyl (DTr) group as an aid in the separation of solid-phase products has been investigated. This substituent, which can be introduced and removed from the 5'-position in a similar manner to the di-p-anisylphenylmethyl group, helps in the purification of deoxypolynucleotides with up to 140 bases. Their retention time was shown to depend not only on the length of the nucleotide chain and alkyl substituent, but also on the elution gradient rate. In oligoribonucleotide synthesis, the DTr group allows the purification of partially protected sequences, which are more stable to enzymatic degradation and, therefore, more suitable for handling and storage.

Precipitation of nucleotides by calcium phosphate.

Earlier it has been shown that nucleic acids of high molecular weight can be be introduced into cells by coprecipitation with calcium phosphate. We have studied the requirements for calcium phosphate coprecipitation of shorter nucleotides. The degree of coprecipitation of dodecanucleotides lacking terminal phosphate varied between 25 and 72%. Tetramers with a 5'-monophosphate were coprecipitated to 29-87% by calcium phosphate. A high content of guanosine residues and an increased number of terminal phosphate groups increased the degree of coprecipitation of nucleotides. The trinucleotide pppA2'p5'A2'p5'A was effectively precipitated by calcium phosphate but the monophosphate and the core structure were not.

Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides.

We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides. It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone. Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable. Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications.

Metal chelate affinity chromatography. I. Influence of various parameters on the retention of nucleotides and related compounds.

The influence of various parameters, such as pH, ionic strength and temperature, on the retention of different nucleotides and related compounds on copper chelate gels has been investigated in order to understand the respective roles played by the different solute constituents (i.e., heterocyclic bases, sugars and phosphate groups) in the interaction and to define optimal conditions for subsequent application to the fractionation of oligo- and polynucleotides.

The separation of nucleic acids and related compounds on nucleobase-coupled cellulose.

The affinity chromatography on uracil-coupled cellulose was carried out for the separation of nucleosides, nucleotides and oligonucleotides. Adenine derivatives exhibited a high affinity to uracil-cellulose, and sequencial isomers of oligonucleotides containing adenine residue were resolved. Poly(A) was strongly bound to uracil-cellulose and recovered by the elution with 7M urea. This procedure was extended to the isolation of mRNA containing poly(A) sequences.

Control of extraction of deoxyribonucleic acid and apurinic acid by polyethylene glycol in Feulgen hydrolysis.

The polymer polyethylene glycol combined with hydrochloric acid was used during the hydrolysis stage of the Feulgen method in order to limit the movement of the soluble molecular fragments developed through hydrolytic breakdown of the deoxyribonucleic acid. The diffusion of the purines was not influenced by the addition of concentration of the polymer up to 30%. On the other hand, a substantial retardation of the extraction of deoxyribonucleic acid and apurinic acid was noted. This indicates that deoxyribonucleic acid and apurinic acid are extracted in chains of considerable length. The decrease in diffusion rate was greater at 1 M HCl than at 0.3 and 6 M HCl, which indicates that the deoxyribonucleic acid-apurinic acid fragments are larger at the medium concentration of acid than at the low and high concentrations. The results thus strengthen the view that deoxyribonucleic acid and apurinic acid are extracted in chains during hydrolysis, and that the critical length at which the chains becomes diffusible is one of the main factors determining the form of the Feulgen hydrolysis curve. The use of polyethylene glycol to extend the maximum of the hydrolysis curve, and thus to make the temperature and time of hydrolysis less critical, is recommended.