Attila Szemzo

Base-modified oligodeoxynucleotides. I. effect of 5-alkyl, 5-(1-alkenyl) and 5-(1-alkynyl) substitution of the pyrimidines on duplex stability and hydrophobicity

Abstract Properties of oligodeoxynucleotides essential for the antisense effect can be favourably modified by substitution of the heterocyclic base moiety. This is shown here for duplex stability by 5 alkyl, 5-(1-alkenyl) and 5-(1-alkynyl) substitution of the pyrimidines in the self-complementary (dT-dA) 10 and (dC-dG) 6 oligomers as well as for the hydrophobic character of the (dT-dA) 10 series.

Acyclic oligonucleotide analogues

Acyclic analogues of oligothymidylate and oligoadenylate and their alternating copolymers were synthesized to study their thermal melting, their stability against snake venom phosphodiesterase and their primer/template properties using the Klenow fragment of the Escherichia coli DNA polymerase I enzyme. Acyclic dodecaadenylate (GlyA)12 hybridized to dodecathymidylate p(dT)12, and the complex presented a sharp melting with a Tm at 24 degrees C. This association was confirmed by circular dichroism curves which were similar to those of the natural oligonucleotide duplexes in A-conformation. (GlyA)12 proved very stable against snake venom phosphodiesterase hydrolysis. The reaction rate was more than 10,000 times slower than that of p(dT)12. (GlyA)12 served as a primer for the Klenow DNA polymerase. When (GlyA)12 was complexed with the poly(dT) template, the enzyme polymerized dATP but the reaction was much slower than with the (GlyT)12 primer. Molecular modelling of atactic (GlyA)12.(dT)12 of the A-conformation indicates that this conformation is energetically possible.

Poly(amino2dA-dT) Isomerizes into the Unusual X-DNA Double Helix at Physiological Conditions Inducing Z-DNA in Poly(dG-methyl5dC)

It is demonstrated that a two-state conformational isomerization is induced in the poly(amino2-dA-dT) duplex by submillimolar concentrations of divalent magnesium cations in low-salt aqueous solution. The isomerization is fast and has a low degree of cooperativity. The resulting conformer is the unusual X-DNA double helix originally observed with poly(dA-dT) at very high concentrations of CsF. Interestingly, the X form is induced in poly(amino2dA-dT) under the physiological conditions when poly(dG-methyl5dC) assumes Z-DNA. The same conditions of stabilization are presumably connected with the fact, observed in previous phosphorus NMR studies, that Z- and X-DNA have similar polydinucleotide backbone architectures. Results presented in this work permit to specify base pair exocyclic groups responsible for the radically different conformational variability of the synthetic DNA molecules containing alternating purine-pyrimidine sequences of GC or AT base pairs.

Application of chromatographic retention data in an investigation of a quantitative structure—nucleotide incorporation rate relationship

Abstract A series of 5-alkyl-, 5-alkenyl- and 5-alkynyl-substituted deoxyuridines and their triphosphate derivatives were synthesized and studied in DNA polymerase reactions. The initial rate of incorporation of the derivatives catalysed by Klenow fragment DNA polymerase enzyme ( E. coli ) was measured. Calf thymus DNA and synthetic poly (dA—dT) served as templates. The rate values were expressed as a percentage relative to the incorporation rate of natural substrate dTTP. The high-performance liquid chromatographic (HPLC) retention behaviours of the nucleoside derivatives were investigated on silica and reversed-phase stationary phases using various mixtures of ethyl acetate—methanol and methanol—water, as respectively, mobile phases. According to the results of principal component analysis, the HPLC retention data describe the hydrophobic properties of the compounds. The inclusion complex stability constants of the derivatives with cyclodextrins determined by reversed-phase thin-layer chromatography served as a measure of the steric properties of the subtituents. The electronic properties of the 5-substituents were characterized by the Swain-Lupton inductive and resonance parameters. The results of the stepwise linear regression analysis of the nucleotide incorporation rate data and the above-mentioned physico-chemical data revealed the importance of the electronic, steric and hydrophobic properties of the substituents in the DNA polymerase reactions. The importance of the steric parameter was more significant when the poly (dA—dT) template was used instead of the random base sequence template (calf thymus DNA).

Destabilization of the duplex and the high-salt Z-form of poly(dG-methyl5dC) by substitution of ethyl for the 5-methyl group.

The B-to-Z conformational transition of poly(dG-dC) is highly promoted by 5-methyl substitution of the dC moiety, i.e. in poly(dG-methyl5dC). By the synthesis of a new poly(dG-dC) analogue, poly(dG-ethyl5dC), the effect of a longer alkyl-chain substituent of dC on structure and conformation has been studied with ultraviolet absorption melting profiles and circular dichroism spectroscopy. The 5-ethyl substituent in poly(dG-ethyl5dC) destabilizes the duplex structure against thermal denaturation compared with both poly(dG-methyl5dC) and poly(dG-dC). C.d. studies also reveal that for the high-salt B-Z transition of poly(dG-ethyl5dC) a higher NaCl concentration is required than for that of poly(dG-methyl5dC), although much lower than for poly(dG-dC). However low-salt Z-DNA in poly(dG-ethyl5dC) shows unique features, e.g. it needs no divalent cations to be stable. The low-salt B-Z transition of poly(dG-ethyl5dC) can also be observed by the absorption-temperature melting profile, in contrast to both poly(dG-methyl5dC) and poly(dG-dC). The effects of MgCl2 concentration, temperature, acid pH and trifluorethanol on the conformation of poly(dG-ethyl5dC) have also been determined.

The Unusual X-Form DNA in Oligodeoxynucleotides: Dependence of Stability on the Base Sequence and Length

X-form is an unusual double helix of DNA adopted by poly(dA-dT) or (dT-dA)4 at high concentrations of CsF. On the other hand, poly(dA), poly(dT), (dA-dT)4 and most other DNAs do not adopt this conformer. Here we demonstrate that the X-form is strongly destabilized by GC pairs or even minute perturbations of the alternating pyrimidinepurine sequence. For example, the 30-mer d(TATAAT)5, containing five tandem repeats of the Pribnow box, fails to isomerize into the X-form. After (dT-dA)4, the 16-mer (dT-dA)8 is shown to be the second most predisposed oligodeoxynucleotide in the (dT-dA)n series to isomerize into the X-form while the duplex lengths corresponding to n = 3,5,6,7,9,12 and 20 make the X-form unstable even in the strictly alternating (dT-dA)n sequence. Consequently, the (dT-dA)n duplex length is also a crucial factor of the X-form stability on the oligodeoxynucleotide level. We discuss a possibility that the X-form is a solution counterpart of the D-form adopted in dehydrated poly(dA-dT) fibers because properties of these two conformers are remarkably similar in many respects.

A study of substrate specificity of mammalian and bacterial DNA polymerases with 5-alkyl-2'-deoxyuridine 5'-triphosphates.

DNA polymerases from procaryotic sources can utilize a variety of dTTP analogues as substrates. We studied here in vitro DNA syntheses catalyzed by DNA polymerase alpha and beta of calf thymus, and for comparison, by the Escherichia coli DNA polymerase I large fragment enzyme in the presence of 5-alkyl derivatives of dUTP as dTTP substrate analogues, using activated DNA as template-primer. The alkyl substituents were n-alkyl (from ethyl to hexyl) and iso-alkyl (isopropyl and tert-butyl) groups. All enzymes were active in the presence of each modified dTTP, incorporation rates of [3H]dAMP or [3H]dGMP were, however, much lower with the analogues than with dTTP. According to relative incorporation rates, alpha-polymerase in DNA synthesis was found to be less sensitive to changes in the length of the alkyl substituent of 5-n-alkyl-dUTPs than beta-polymerase or the E. coli enzyme. Evidence for the incorporation of the analogues was presented for 5-[2-14C]isopropyl-dUTP.

Some biochemical properties of an acyclic oligonucleotide analogue. A plausible ancestor of the DNA

As acyclic oligonucleotides have been suggested as a primitive model of DNA or RNA in prebiotic times, we compared some biochemical properties of these analogues to that of natural ones. Firstly, an acyclic analogue of deoxyribonucleoside triphosphates was tested as a potential substrate of enzymes intervening in nucleic acids synthesis. GlyTTP, a dTTP analogue with a missing 2′-methylene group is notaccepted as a substrate by either DNA polymerase or deoxynucleotidyl terminal transferase (TdT). Secondly, themodified dodecathymidylate (GlyT)12, the racemic acyclic sugar analogue of (dT)12, proved to be anefficient primer for DNA polymerase and TdT, though the associative properties of (GlyT)12 are very weak as shown by UV spectroscopy in phosphate buffer without magnesium chloride. But (GlyT)12 has the advantage to be 500-times more stable against hydrolysis by snake venom phosphodiesterase than the corresponding oligothymidylate.

Inhibition of IgE production by epsilon (ϵ) chain-specific antisense oligonucleotides (AOs) studied on human myeloma cell line U266 and peripheral blood mononuclear cells of a patient with hypereosinophilia

Based on cDNA sequence data epsilon chain-specific antisense oligonucleotides were synthesized and checked on in vitro IgE production. Using peripheral blood cells from a hypereosinophilic patient and a human IgE myeloma cell line, U266, marked reduction of in vitro IgE production measured by PRIST was observed. The effect of epsilon antisenses proved to be isotype specific since IgG production by both peripheral blood cells and a lymphoma cell line, CESS, was not affected. Moreover, the expression of other markers on U266 (interleukin-6 receptor and gp130) were not influenced by epsilon-specific antisense oligonucleotides.