The affinity of an oligodeoxynucleotide-peptide conjugate for an RNA hairpin loop depends on stereochemistry at the DNA-peptide junction.

Molecular models of an oligodeoxynucleotide-peptide conjugate complexed to an RNA hairpin loop were constructed to assess the effect of stereoisomerism at the point of attachment of the peptide to the oligodeoxynucleotide on the affinity of the conjugate for an RNA target. The peptide portion of the oligodeoxynucleotide-peptide conjugate, (L-lysine)8, was covalently attached to the N-allyl group of (D)- or (L)-aspartic alcohol that was incorporated into the interior of an antisense oligodeoxynucleotide. The stereocenter in the oligodeoxynucleotide interior originates from either (D)- or (L)-aspartic alcohol. The oligodeoxynucleotide portion of the oligodeoxynucleotide-peptide conjugate forms Watson-Crick base pairs with the single-stranded RNA that flanks the RNA hairpin loop. The positively charged peptide makes specific electrostatic contacts with the negatively charged phosphate backbone of the RNA hairpin loop when attached to the N-allyl of (D)-aspartic alcohol but does not have the proper orientation to make these electrostatic contacts when attached to the N-allyl of (L)-aspartic alcohol. This modelling study emphasizes the importance of stereocontrol at the point of branching in synthesizing oligodeoxynucleotide-peptide conjugates for binding of RNA hairpin loops.

The 5' stem-loop from human U4 snRNA adopts a novel conformation stabilized by G-C and G-U base pairs.

1H NMR and molecular modeling studies of the 5' stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5' side and a single bulged A on the 3' side. Both stems also had bulged A residues. 1H NMR experiments performed on solutions of the 35 mer stem-loop, the stem region, and the loop region confirmed the 35 mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.

Solution structures of 5-fluorouracil-substituted RNA duplexes containing G-U wobble base pairs.

The structures and stabilities of three RNA duplexes that differed only in the position of 5-fluorouridine (FUrd) substitution were elucidated using NMR spectroscopy and UV hyperchromicity studies to determine if FUrd substitution altered the structure or stability of RNA duplexes that contained G-U base pairs. The duplexes investigated corresponded to the region of the U4-U6 snRNA complex that contained the 5' terminus of U4 snRNA. The control duplex contained a G-U wobble base pair and also a G-A mismatched base pair. FUrd was substituted in one duplex at the G-U wobble base pair and in the second duplex at an A-U base pair adjacent to the wobble base pair. FUrd substitution slightly destabilized the duplex that contained a G-FU base pair but stabilized the duplex that contained an A-FU base pair. NOESY spectra were used to determine interproton distances, and these distance constraints were used in a restrained molecular dynamics protocol to determine the three-dimensional structures of these RNA duplexes. Analyses of helical parameters, backbone torsion angles, and rms deviations between the final structures revealed no systematic differences due to FUrd substitution in RNA duplexes that contained G-U base pairs. The G-FU base pair adopted wobble geometry, while the G-A mismatch formed a sheared base pair. NOESY spectra in H2O solution revealed the imino 1H from FUrd exchanged more rapidly with solvent than did the Urd imino 1H but did not show the G-FU base pair adopted an ionized structure. Reduced stacking occurred for the G-FU base pair relative to the G-U base pair in the time-averaged structure, and this, rather than ionization of the base pair, was responsible for the slight destabilization of the duplex that contained the G-FU base pair.

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.

Solution structures of 5-fluorouracil-substituted DNA and RNA decamer duplexes.

The structures in solution of eight oligonucleotide duplexes each containing either zero, one, or two 5-fluorodeoxyuridine (FdUrd) or 5-fluorouridine (FUrd) nucleosides were determined by the combined use of NMR spectroscopy, restrained molecular dynamics, and full relaxation matrix refinement to determine how FdUrd and FUrd substitution affects the structure of duplex DNA and RNA and to establish whether structural differences due to FdUrd and FUrd substitution in nucleic acids may be responsible, in part, for the biological effects of the anticancer drug 5-fluorouracil (FUra). The nucleic acid directed effects of FUra include induction of single-strand breaks in duplex DNA and altered processing of pre-mRNA and rRNA. Four self-complementary oligodeoxyribonucleotide sequences were prepared and studied as duplexes in aqueous solution: (5' dGCGAAUUCGC)2, (5' dGCGAAUFCGC)2, (5' dGCGAAFUCGC)2, and (5' dGCGAAFFCGC)2. The corresponding oligoribonucleotide sequences (5' rGCGAAUUCGC)2, (5' rGCGAAUFCGC)2, (5' rGCGAAFUCGC)2, and (5' rGCGAAFFCGC)2 were also prepared and studied. The helical parameters for the structures of these eight duplexes were analyzed to determine how substitution of FdUrd and FUrd affects the three-dimensional structures of duplex DNA and RNA. FdUrd substitution affects the base roll angle at the site of FdUrd substitution, causing the helical axis of FdUrd-substituted DNA duplexes to be bent compared to the nonsubstituted duplex. A-FUrd base pairs show substantial RMS deviations from A-Urd base pairs in all three of the RNA duplexes substituted with FUrd. Bending of the helical axis due to FdUrd substitution may contribute to the occurrence of single-strand breaks in duplex DNA while the altered structures of A-FUrd base pairs may affect RNA-RNA and RNA-protein recognition.

Homology modeling of an RNP domain from a human RNA-binding protein: Homology-constrained energy optimization provides a criterion for distinguishing potential sequence alignments.

We have recently described an automated approach for homology modeling using restrained molecular dynamics and simulated annealing procedures (Li et al, Protein Sci., 6:956-970,1997). We have employed this approach for constructing a homology model of the putative RNA-binding domain of the human RNA-binding protein with multiple splice sites (RBP-MS). The regions of RBP-MS which are homologous to the template protein snRNP U1A were constrained by "homology distance constraints," while the conformation of the non-homologous regions were defined only by a potential energy function. A full energy function without explicit solvent was employed to ensure that the calculated structures have good conformational energies and are physically reasonable. The effects of mis-alignment of the unknown and the template sequences were also explored in order to determine the feasibility of this homology modeling method for distinguishing possible sequence alignments based on considerations of the resulting conformational energies of modeled structures. Differences in the alignments of the unknown and the template sequences result in significant differences in the conformational energies of the calculated homology models. These results suggest that conformational energies and residual constraint violations in these homology-constrained simulated annealing calculations can be used as criteria to distinguish between correct and incorrect sequence alignments and chain folds.