Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond

Single-stranded regions in RNA secondary structure are important for RNA–RNA and RNA–protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals ‘well-determined’ single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit β-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA–RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C- and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and 15N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional [15N] 1H heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 microM concentration range, with a total of 30 nmol or approximately 40 micrograms of RNA in approximately 150 microliters, give strong NMR signals in a short accumulation time. The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems. This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

A secondary-structure model for the self-cleaving region of Neurospora VS RNA.

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.

Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon

The Bacillus subtilis pyr operon is regulated by exogenous pyrimidines by a transcriptional attenuation mechanism. Transcription in vitro from pyr DNA templates specifying attenuation regions yielded terminated and read-through transcripts of the expected lengths. Addition of the PyrR regulatory protein plus UMP led to greatly increased termination. Synthetic antisense deoxyoligonucleotides were used to probe possible secondary structures in the pyr mRNA that were proposed to play roles in controlling attenuation. Oligonucleotides predicted to disrupt terminator structures suppressed termination, whereas oligonucleotides predicted to disrupt the stem of antiterminator stem-loops strongly promoted termination at the usual termination site. Oligonucleotides that disrupt a previously unrecognized stem-loop structure, called the anti-antiterminator, the formation of which interferes with formation of the downstream antiterminator, suppressed termination. We propose that transcriptional attenuation of the pyr operon is governed by switching between alternative antiterminator versus anti-antiterminator plus terminator structures, and that PyrR acts by UMP-dependent binding to and stabilization of the anti-antiterminator.

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra- and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stem-loops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.

Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

A physical basis for protein secondary structure

A physical theory of protein secondary structure is proposed and tested by performing exceedingly simple Monte Carlo simulations. In essence, secondary structure propensities are predominantly a consequence of two competing local effects, one favoring hydrogen bond formation in helices and turns, the other opposing the attendant reduction in sidechain conformational entropy on helix and turn formation. These sequence specific biases are densely dispersed throughout the unfolded polypeptide chain, where they serve to preorganize the folding process and largely, but imperfectly, anticipate the native secondary structure.

Conserved stem–loop structures in the HIV-1 RNA region containing the A3 3′ splice site and its cis-regulatory element: possible involvement in RNA splicing

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3′ splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem–loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem–loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3′ A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

Fourier Transform Infrared Microspectroscopy Detects Changes in Protein Secondary Structure Associated with Desiccation Tolerance in Developing Maize Embryos1

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of α-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the α-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of α-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.

Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.

Rational engineering of enzymes involves introducing key amino acids guided by a knowledge of protein structure to effect a desirable change in function. To date, all successful attempts to change specificity have been limited to substituting individual amino acids within a protein fold. However, the infant field of protein engineering will only reach maturity when changes in function can be generated by rationally engineering secondary structures. Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to systematically invert the coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant, which is twice as active as wild type, contains four amino acid substitutions and an alpha-helix and loop that replaces the original beta-turn. These results demonstrate that rational engineering of secondary structures to produce enzymes with novel properties is feasible.

Lyophilization-induced reversible changes in the secondary structure of proteins.

Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fourier-transform infrared spectroscopy in the amide III band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the beta-sheet content and lowers the alpha-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Maintenance of pre-mRNA secondary structure by epistatic selection.

Linkage disequilibrium between polymorphisms in a natural population may result from various evolutionary forces, including random genetic drift due to sampling of gametes during reproduction, restricted migration between subpopulations in a subdivided population, or epistatic selection. In this report, we present evidence that the majority of significant linkage disequilibria observed in introns of the alcohol dehydrogenase locus (Adh) of Drosophila pseudoobscura are due to epistatic selection maintaining secondary structure of precursor mRNA (pre-mRNA). Based on phylogenetic-comparative analysis and a likelihood approach, we propose secondary structure models of Adh pre-mRNA for the regions of the adult intron and intron 2 where clustering of linkage disequilibria has been observed. Furthermore, we applied the likelihood ratio test to the phylogenetically predicted secondary structure in intron 1. In contrast to the other two structures, polymorphisms associated with the more conserved stem-loop structure of intron 1 are in low frequency, and linkage disequilibria have not been observed. These findings are qualitatively consistent with a model of compensatory fitness interactions. This model assumes that mutations disrupting pairing in a secondary structural element are individually deleterious if they destabilize a functionally important structure; a second "compensatory" mutation, however, may restabilize the structure and restore fitness.