Thomas Neilson

Improved free energies for G · C base-pairs☆

Thermodynamic parameters of helix formation are reported for seven oligoribonucleotides containing only G.C pairs. These data are used with the nearest-neighbor model to calculate enthalpies and free energies of base-pair formation for G.C pairs. For helix initiation, the free energy change at 37 degrees C, delta G(0)37, is +3.9 kcal/mol; for helix propagation, the delta G(0)37 values are -2.3, -3.2 and -3.3 kcal/mol for C-G, G-G and G-C neighbors, respectively.

Z-RNA: A Left-Handed Double Helix

The synthetic polyribonucleotide, poly [r(G-C)], has been shown to undergo a transition from the right handed A-form to a left-handed Z-conformation.1 This is the first major conformational change found in double-stranded RNA.2 Therefore it is important to corroborate this observation, to characterize the conditions that produce Z-RNA, and to determine some of the properties of this novel left-handed structure. This article is a summary of our results in this area.

Invariant adenosine residues stabilize tRNA D stems

Inspection of the sequences of reported tRNAs [l] reveals 2 adenosine residues that nearly always (166 out of 177 cases) occupy positions 14 and 21 at the D-loop-stem junction. Apparently, the nature of these residues has been conserved and perhaps they are involved in some tRNA function, for example, aminoacyl-tRNA synthetase recognition [2,3]. Their presence may also satisfy a structural aspect controlling native conformation, namely an invariant A . U base pair [4,5]. This report resolves the controversy by proposing that the invariant adenosines stabilize D-stem duplexes, features of secondary cloverleaf structure [6]. D-Stems contain only 3 or 4 WatsonCrick base pairs while other stem duplexes have 5 or more. Melting studies have shown D-stems to be the least stable regions in tRNAs [7]. Three-dimensional tertiary structure of yeast tRNA [4] shows adenosine residues 14 and 21 (see fig.1) to be coplanar, and each base-stacked (a vertical electronic interaction between aromatic rings) to the adjacent D-stem duplex [5]. Steric tolerance exists, however, since the adenosine at position 14 is displaced within the helix as a result of its participation in a tertiary Sobell-type A . U base pair [8] with an invariant uridine residue at position 8. This displacement from the normal RNA-A helical geometry at the ends of a duplex can be considered as partial strand unwinding. Extension of base stacking to the invariant adenosine residues, is still possible, and therefore enhances overall D-stem stability.

Contribution of a G·U base pair to the stability of a short RNA helix

Variable temperature 1H n.m.r. spectroscopic studies have shown that the contribution of an internal G·U base pair approximates that of an A·U base pair in the stability of a short RNA helix.

Improved Parameters for Prediction of RNA Secondary Structure and Insights into why RNA forms Double Helixes

Thermodynamic parameters for double helix formation have been measured for a large number of oligoribonucleotides. These data have been analyzed to provide free energy changes associated with base pairs, dangling ends, and base mismatches. The results suggest base stacking and base pairing are important determinants of RNA stability, but that hydrophobic bonding is not. The improved thermodynamic parameters are applied to predict secondary structures for the self splicing intervening sequence from the ribosomal RNA precursor of Tetrahymena thermophila.

1H-Nuclear magnetic resonance of intercalators and rGCA: A potential mutagenicity probe

Variable temperature 1H-nuclear magnetic resonance (NMR) has been used to study the interaction of the RNA trimer, GpCpA, with the intercalators ethidium bromide and the acridine derivatives; proflavin, 9-amino-acridine, acridine orange, acridine yellow and acriflavin. The complexes formed were studied at nucleic acid to drug ratios of 1:1 and 5:1, the latter being useful in defining the effects of structural variation in the acridine series and in determining the site of intercalation. All the intercalators greatly stabilized the oligonucleotide duplex, the average melting temperature (Tm) increasing by up to 30 degrees C. Significant changes in individual Tms and chemical shifts were observed for all the GpCpA protons. 9-Amino-acridine and acriflavin did not stabilize the GpCpA duplex as substantially as the other acridine derivatives. It is suggested that this intercalator:GpCpA system, and its associated NMR-derived Tm, is a useful physical probe for potential mutagens.

Parameters for proton chemical shift calculation in oligoribonucleotides

A Set of parameters has been developed that provides a new mathematical approach for the assignment of the 1H n.m.r. chemical shifts for heterobase ring and anomeric protons in oligoribonucleotides.

Sequencing of short RNA oligomers by proton nuclear magnetic resonance

Advances have been made in the sequencing of RNA by enzymic [l-3] and chemical [4,5] methods. A limiting feature of these procedures is the quantity of RNA available, unfortunately 2-3 orders of magnitude less than for DNA sequencing [6,7]. These methods can also be unreliable when applied to short RNA fragments, especially sequences containing stretches of guanosine residues where resolution in polyacrylamide gel analysis is ambiguous. DNA sequencing suffers the same drawback [6]. More facile RNA duplex formation [ 81 creates problems for base-specific endonuclease digestions which are more efficient on single-stranded RNA. Lack of RNA restriction enzymes can be circumvented by recourse to short complementary DNA sequences and the use of ribonuclease H [9] which cleaves within or adjacent to the hybridization site. However, existing RNA secondary structure can inhibit expected RNA:DNA duplex formation and a selection of short DNA sequences must be assayed to achieve a constant fragment pattern. tation, in particular, the availability of very high field magnets and more rapid scanning procedures, will dramatically reduce the sample size in the near future. However, the present sample size is convenient for checking oligomers from chemical synthesis. The procedure is based on the fact that the resonances of the nonexchangeable aromatic and anomeric protons of adenosine, guanosine, cytidine and uridine exhibit changes in their particular chemical shifts which depend on the bases immediately surrounding a particular residue in a given sequence. These sequence effects on chemical shifts of protons on a particular residue do not extent significantly beyond the nextnearest neighbour at 70°C [ IO,1 I]. A set of ‘H NMR chemical shift parameters [ 121 (derived from the NMR spectra of trinucleoside diphosphates) is available which allows the chemical shifts of protons in longer sequences to be calculated. Consequently, the base sequence in longer oligoribonucleotides can be determined.