Protonated polynucleotide structures

The circular dichroism spectra of poly(dG). poly(dC) have been studied as a function of pH in 0.15 M NaCl solution. Acid titration to pH 2.5 showed two transitions, one around pH 5, the second below pH 3. These transitions are disproportionation reactions and are not due to the dissociation of the complexes. Alkaline back titration to neutrality showed only one step above pH 7, without appearance of the intermediate form. The hysteresis loop observed gave rise to a metastable (probably protonated) form at pH 7 which reverted to the neutral form upon heating or alkali treatment. In order to obtain again the metastable hysteretic form, a whole titration cycle to pH 2.5 had to be performed.Abbreviation CD circular dichroism Dedicated to Prof. A Janke (Vienna) on his 85th birthday.Part 13 is ref. [1].     

Mechanism of polynucleotide phosphorylase

The de novo polymerization of RNA initiated by polynucleotide phosphorylase from nucleoside diphosphates was examined. End group analysis performed under conditions designed to specifically end label the polymer revealed no evidence for a 5'-pyrophosphate-terminated polymer. However, we observed preferential incorporation of the ADP alpha S(RP) diastereomer into the 5' end (Marlier & Benkovic, 1982) in chain initiation, suggesting that the enzyme incorporates a nucleoside diphosphate specifically into the 5' end of the product, with subsequent enzymatic removal of the polyphosphate linkage. No evidence could be obtained for a covalent adduct between the enzyme and the 5' end of the polymer chain, despite the high processivity of the polymerization reaction. Gel electrophoretic analysis showed the polymer to be highly disperse, varying from 1 to 30 kb. Scanning transmission electron microscopy supported this product analysis and further suggested that (i) each subunit can produce an RNA polymer and (ii) both 5' and 3' ends of the RNA can be bound simultaneously to the same or differing enzyme molecules.     

Animal tissue polynucleotide phosphorylase]

Localization, physico-chemical and catalytic properties and possible biological functions of polynucleotide phosphorylase (PNPase) from animal tissues are discussed. In animal tissue cells PNPase has multiple localization; the major amount of the enzyme is localized in the endoplasmic reticulum ribosomes. In the nuclei PNPase, similar to other endo- and exo-RNAses participates in the processing of precursor molecules of mature forms of RNA, whereas in the cytoplasm it is involved in the destruction of polyribosomes in the polyribosomes of rapidly growing tissues the activity of PNPase is extremely decreased. The mechanisms regulating the PNPase activity in rapidly growing tissues are discussed.     

Nanoparticulate systems for polynucleotide delivery

Nanotechnology has tremendously influenced gene therapy research in recent years. Nanometer-size systems have been extensively investigated for delivering genes at both local and systemic levels. These systems offer several advantages in terms of tissue penetrability, cellular uptake, systemic circulation, and cell targeting as compared to larger systems. They can protect the polynucleotide from a variety of degradative and destabilizing factors and enhance delivery efficiency to the cells. A variety of polymeric and non-polymeric nanoparticles have been investigated in an effort to maximize the delivery efficiency while minimizing the toxic effects. This article provides a review on the most commonly used nanoparticulate systems for gene delivery. We have discussed frequently used polymers, such as, polyethyleneimine, poly (lactide-co-glycolide), chitosan, as well as non-polymeric materials such as cationic lipids and metallic nanoparticles. The advantages and limitations of each system have been elaborated.     

New wrinkles on polynucleotide duplexes

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As4T):poly d(As4T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c = 7.40nm, gamma = 90 degrees. The c dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4(3) helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain C3'-endo furanose rings and the pyrimidine nucleotides C2'-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.     

Fuzzy polynucleotide spaces and metrics

The study of genetic sequences is of great importance in biology and medicine. Mathematics is playing an important role in the study of genetic sequences and, generally, in bioinformatics. In this paper, we extend the work concerning the Fuzzy Polynuclotide Space (FPS) introduced in Torres, A., Nieto, J.J., 2003. The fuzzy polynuclotide Space: Basic properties. Bioinformatics 19(5); 587–592 and Nieto, J.J., Torres, A., Vazquez-Trasande, M.M. 2003. A metric space to study differences between polynucleotides. Appl. Math. Lett. 27:1289–1294: by studying distances between nucleotides and some complete genomes using several metrics. We also present new results concerning the notions of similarity, difference and equality between polynucleotides. The results are encouraging since they demonstrate how the notions of distance and similarity between polynucleotides in the FPS can be employed in the analysis of genetic material.     

Sequence studies of nonradioactive Mycoplasma tRNAPhe with the aid of polynucleotide phosphorylase and polynucleotide kinase

The known methods of enzymatic phosphorylation with [(32)P]phosphate of the 3'- or 5'-hydroxyl group of an oligonucleotide have been applied to oligonucleotides derived from Mycoplasma tRNA(Phe). The fingerprints obtained by both methods are very similar to each other and to that of uniformly labelled tRNA. The sequence of some oligonucleotides was determined by partial digestion of the 3'-phosphorylated fragment with spleen phosphodiesterase and of the corresponding 5'-phosphorylated fragment with venom phosphodiesterase.     

Template-directed polynucleotide synthesis on mineral surfaces

Ferric hydroxide, a plausible prebiotic material, strongly adsorbs polynucleotides. We show that adsorption on ferric hydroxide and on several other minerals has no effect, under the conditions studied, on the template-directed oligomerization of guanylic acid on polycytidylic acid.