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The capability of translating capped mRNA is reduced by the human rhinovirus and the mammalian enterovirus. The infection allows translation of uncapped viral RNA. The cleavage of the eukaryotic initiation factor 4-gamma is induced by the viral proteinase 2A. The soluble form of Escherichia coli provides an expression for the nature proteinases 2A of human rhinovirus 2 and coxsackievirus B4.

DNA triple helices offer exciting perspectives toward oligonucleotide-directed control of gene expression. Oligonucleotide analogues are routinely used with modifications in either the backbone or the bases to form more stable triple-helical structures or to prevent their degradation in cells. In this article, different chemical modifications are tested in a model system, which sets up a competition between the purine and pyrimidine motifs. For most modifications, the DeltaH degrees of purine triplex formation is close to zero, implying a nearly temperature-independent affinity constant. In contrast, the pyrimidine triplex is strongly favored at lower temperatures. The stabilization induced by modifications previously known to be favorable to the pyrimidine motif was quantified. Interestingly, modifications favorable to the GT motif (propynyl-U and dU replacing T) were also discovered. In a system where two third strands compete for triplex formation, replacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This purine-to-pyrimidine triplex conversion depends on the chemical nature of the triplex-forming strands and the stability of the corresponding triplexes.

Inosine has the ability to act as a "wild-card" binding nonspecifically to both A.T and G.C base pairs. This has obvious implications for the design of oligonucleotide site-directed probes. In this paper we present a series of oligonucleotides with a 5'pur9-pyr9-pyr9 motif which are designed to fold up sequentially into intramolecular triple helices. One or more inosines are incorporated into the Hoogsteen strands in place of T's and/or C's. Once folded into the triplex, the inosine-containing third strand is incorporated in parallel orientation to the purine strand of the duplex. The influence of inosine on the triplex-duplex equilibrium, characterized by the melting temperature (Tm) and on the phase boundaries, as a function of pH and/or ionic strength, has been assessed by means of UV and CD spectroscopy. There are two distinguishable influences of third-strand inosines which affect binding, namely, backbone distortion due to bulkiness (I for T and I for C+) and/or loss of intramolecular ion pairs between protonated cytosines and the backbone phosphates (I for C+). A single thymine replacement drops the Tm by 25.0 (+/- 2.1) degrees C, and replacing a single protonated cytosine drops the Tm by 32.1 (+/- 1.0) degrees C at pH 6.0. On introducing two inosines in place of thymines, the Tm at pH 6.0 of the triple helix to hairpin transition is lowered by 35.5 (+/- 1.4) degrees C; on introducing two inosines in place of cytosines, the Tm drops by 44.5 (+/- 1.0) degree C, and on replacing a cytosine and a neighboring thymine with inosines, the Tm of the same transition is lowered by 29.2 (+/- 1.6) degrees C. Replacing more than two thymines or cytosines, respectively, eliminates the binding of the Hoogsteen strand at room temperature altogether. Under no circumstances does inosine replacement stabilize the triplex helix: it is a poor substitute and its role as a wild-card is limited.

Electrostatic effects dominate many aspects of nucleic acid behavior in a sequence independent manner. Sequence dependent electrostatic effects are introduced when a polypyrimidine, which contains one or more protonated cytosines, binds in the major groove (Hoogsteen side) of a complementary Watson-Crick double helix. Depending on the number of cytosines in the third strand (global effect) and on their relative position (local effect), the cytosines either enhance or decrease the binding affinity of the third strand, because adjacent protonated cytosines destabilize the third strand binding compared to cytosines separated by intervening thymines. This local effect (crowding) can reverse the effect of global composition. To investigate the extent of the local and global electrostatic effects further, two families of oligonucleotides have been synthesized. They share as a common design feature that they all fold sequentially into isosterical intramolecular triple helices by way of hairpin intermediates. This is confirmed by P1 nuclease probing, CD spectroscopy, and UV spectroscopy. The thermal stability of these conformations depends on the sequences, pH, and the ionic strength and can be summarized as follows: The energy of third strand binding depends on the protonated cytosine content in the Hoogsteen strand. It increases with increasing cytosine content (global composition) below pH 7.1 (150 mM Na+), decreases above pH 7.1, and is independent of the cytosine content at pH 7.1. At pH 6.75 the energy of binding increases with increasing cytosine content below 400 mM Na+, decreases above 400 mM Na+, and is independent of the global composition at 400 mM Na+.

Previously (Dolinnaya & Fresco, 1992), on the basis of an analysis of UV absorption and CD properties as a function of temperature and pH, the secondary structure of the deoxyoligonucleotide d(A(+)-G)10 was hypothesized to be helical and intramolecular in origin, being stabilized not by stacked bases or hydrogen-bonded base pairs but instead by ionic bonds between positively charged adenine residues and distal negatively charged phosphates. Several other properties are now shown to be consistent with this unusual type of structure. The molecular weight determined for d(A(+)-G)10 by sedimentation equilibrium is that of the single strand, and consistent with this, there is no molecular weight change on helix disruption. Formation of the d(A(+)-G)10 helix is accompanied by cooperative uptake of nine protons, corresponding to nine adenine residues that can form ionic bonds with all the available distal phosphates, i.e., the n+1 or the n+2 phosphates. The thermodynamic parameters of this helical structure obtained from both van't Hoff analysis of the melting of the structure and calorimetric measurements are in keeping with the ionic properties of the proposed structure. So are the dependence of its stability on pH and ionic strength, and also on oligomer length when compared with the behavior of d(A(+)-G)6. The possible role of this type of secondary structure in protein recognition of the single-stranded homopurine element of H-DNA is evaluated.

Ultraviolet spectroscopic and nuclear magnetic resonance (NMR) studies have shown that poly[r(G-C)] in a solution of 4 M NaClO4 undergoes a transition to a left-handed Z-RNA helix upon raising the temperature to 60 degrees C [Hall, K., Cruz, P., Tinoco, I., Jr., Jovin, T. M., & van de Sande, J. H. (1984) Nature (London) 311, 584-586]. In the present report, the transition temperature of this particular order/order transition is shown to increase with decreasing NaClO4 concentration to about 110 degrees C, above which only the helix-to-random coil transition is detectable. The reversibility and cooperativity of the helix/helix conversion has facilitated the quantitative evaluation of the transition enthalpy by means of differential scanning microcalorimetry. In 5 M NaClO4, the transition temperature is 43 degrees C, the conversion enthalpy 4.2 kJ (1.0 kcal) per mole of base pair, and the corresponding entropy change 13 J (3.1 cal) deg-1. The van't Hoff enthalpy for the same process, determined from the temperature dependence of the optical transition, is 0.26 MJ (62 kcal) per mole of cooperative unit. The ratio of the two enthalpy values yields an apparent cooperative length for the A-Z transition of poly[r(G-C)] of approximately 60 base pairs, indicative of a concerted all-or-none process.