Montmorillonite,Oligonucleotides, RNA and Origin of Life

Na-montmorillonite prepared from Volclay by the titration method facilitates the self-condensation of ImpA, the 5'-phosphorimidazolide derivative of adenosine. As was shown by AE-HPLC analysis and selective enzymatic hydrolysis of products, oligo(A)s formed in this reaction are 10 monomer units long and contain 67% 3',5'-phosphodiester bonds (Ferris and Ertem, 1992a). Under the same reaction conditions, 5'-phosphorimidazolide derivatives of cytidine, uridine and guanosine also undergo self-condensation producing oligomers containing up to 12-14 monomer units for oligo(C)s to 6 monomer units for oligo(G)s. In oligo(C)s and oligo(U)s, 75-80% of the monomers are linked by 2',5'-phosphodiester bonds. Hexamer and higher oligomers isolated from synthetic oligo(C)s formed by montmorillonite catalysis, which contain both 3',5'- and 2',5'-linkages, serve as catalysts for the non-enzymatic template directed synthesis of oligo(G)s from activated monomer 2-MeImpG, guanosine 5'-phospho-2-methylimidazolide (Ertem and Ferris, 1996). Pentamer and higher oligomers containing exclusively 2',5'-linkages, which were isolated from the synthetic oligo(C)s, also serve as templates and produce oligo(G)s with both 2',5'- and 3',5'-phosphodiester bonds. Kinetic studies on montmorillonite catalyzed elongation rates of oligomers using the computer program SIMFIT demonstrated that the rate constants for the formation of oligo(A)s increased in the order of 2-mer < 3-mer < 4-mer ... < 7-mer (Kawamura and Ferris, 1994). A decameric primer, dA(pdA)8pA bound to montmorillonite was elongated to contain up to 50 monomer units by daily addition of activated monomer ImpA to the reaction mixture (Ferris, Hill and Orgel, 1996). Analysis of dimer fractions formed in the montmorillonite catalyzed reaction of binary and quaternary mixtures of ImpA, ImpC, 2-MeImpG and ImpU suggested that only a limited number of oligomers could have formed on the primitive Earth rather than equal amounts of all possible isomers (Ertem and Ferris, 2000). Formation of phosphodiester bonds between mononucleotides by montmorillonite catalysis is a fascinating discovery, and a significant step forward in efforts to find out how the first RNA-like oligomers might have formed in the course of chemical evolution. However, as has been pointed out in several publications, these systems should be regarded as models rather than a literal representation of prebiotic chemistry (Orgel, 1998; Joyce and Orgel, 1999; Schwartz, 1999).     

Characteristics of the interactions of cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I complexes with eukaryotic DNA

Primary hepatocyte culture has been used to demonstrate that the cortisol-apolipoprotein A-I complex does not affect the DNA and protein biosynthesis rates, whereas the tetrahydrocortisol-apolipoprotein A-I complex (THC-apoA-I) substantially increases the rates of ³H-thymidine and ¹⁴C-leucine incorporation into DNA and protein, respectively. Small-angle X-ray scattering data show that only THC-apoA-I effectively interacts with eukaryotic DNA, which is accompanied by local DNA melting. The (GCC)_n repeat, a component of many human and other eukaryotic genes, is the most probable region of the interaction of this complex with DNA. An oligonucleotide (duplex) of this type has been synthesized. Its interaction with THC-apoA-I yields a larger complex, which breaks up, giving rise to complementary oligonucleotide strands. They also interact with THC-apoA-I. The equilibrium kinetics of this multiphase process is described. The interaction of the cortisol-apolipoprotein A-I complex with the duplex is less specific and does not cause its breakup or the formation of complementary oligonucleotides.     

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry,circular dichroism and atomic force microscopy

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1–5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K⁺, the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the T_m of the DNA–polyamine complexes obviously increased both in the absence and presence of K⁺. The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.     

Effect of polyisobutylcyanoacrylate nanoparticles and Lipofectin® loaded with oligonucleotides on cell viability and PKCα neosynthesis in HepG2 cells

The aim of the present study was to evaluate the inhibitory effect on protein kinase Ca (PKCα) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKCα antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin® was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKCα neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin® were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKCa neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin®). This non-specific effect was only observed in the phase of PKCα neosynthesis when the cells were first depleted in PKCa by phorbol 12-myristate β-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin®, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.