Removal of psoralen monoadducts and crosslinks by human cell free extracts.

Human cell free extracts are capable of carrying out damage-induced DNA synthesis in response to DNA damage by UV, psoralen, and cisplatin. We show that this damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is 'repair synthesis' and not an aberrant DNA synthesis reaction potentiated by DNA deformed by adducts. By comparing the denaturable fraction of psoralen adducted DNA which becomes labeled in the repair reaction to that of terminally labeled DNA (without repair) we have found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach we have obtained preliminary evidence that this in vitro system is capable of removing psoralen crosslinks as well.     

Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms

Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells.     

Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen–modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex ~3 bases from the 3′-side and ~9 bases from the 5′-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.     

The secondary structure of a messenger RNA precursor probed with psoralen is melted in an in vitro splicing reaction.

The secondary structure of the SP6/mouse insulin precursor RNA was determined by psoralen cross-linking experiments. A series of long-range contacts occur within the left half of the pre-mRNA that contains the intervening sequence. Multiple secondary structures for the pre-mRNA exist since some of the interactions share common sites. In splicing buffer but without the splicing extract added, many of these interactions are stable up to at least 50 degrees C. These interactions, however, are dissociated during the in vitro splicing reaction. This dissociation requires ATP and it occurs during the first 30 min. of the splicing reaction. Pre-mRNAs containing psoralen cross-links in different locations within the RNA molecule were purified and used as substrates for in vitro splicing. Psoralen cross-links at any of the double-stranded regions resulted in complete inhibition of the splicing reaction. This indicates that destabilization of the secondary structure of the SP6/mouse insulin pre-mRNA is necessary for in vitro splicing.     

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.     

A conserved secondary structure for telomerase RNA.

The RNA moiety of the ribonucleoprotein enzyme telomerase contains the template for telomeric DNA synthesis. We present a secondary structure model for telomerase RNA, derived by a phylogenetic comparative analysis of telomerase RNAs from seven tetrahymenine ciliates. The telomerase RNA genes from Tetrahymena malaccensis, T. pyriformis, T. hyperangularis, T. pigmentosa, T. hegewishii, and Glaucoma chattoni were cloned, sequenced, and compared with the previously cloned RNA gene from T. thermophila and with each other. To define secondary structures of these RNAs, homologous complementary sequences were identified by the occurrence of covariation among putative base pairs. Although their primary sequences have diverged rapidly overall, a strikingly conserved secondary structure was identified for all these telomerase RNAs. Short regions of nucleotide conservation include a block of 22 totally conserved nucleotides that contains the telomeric templating region.     

Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion

Psoralen intercalates into double-stranded DNA and photoreacts mainly with thymines to form monoadducts and interstrand cross-links. We used an oligonucleotide model to demonstrate a novel mechanism: the reversal of psoralen cross-links by base-catalyzed rearrangement at 90 degrees C (BCR). The BCR reaction is more efficient than the photoreversal reaction. We show that the BCR occurs predominantly on the furan side of a psoralen cross-link. The cleavage does not result in the breaking of the DNA backbone, and the thymine base freed from the cross-link by the cleavage reaction appears to be unmodified. Similarly, BCR of the furan-side monoadduct of psoralen removed the psoralen molecule and regenerated the unaltered native oligonucleotide. The pyrone-side psoralen monoadduct is relatively resistant to BCR. One can use BCR to perform efficient oligonucleotide-directed, site-specific delivery of a psoralen monoadduct. As a demonstration of this approach, we have hybridized a 19 base long oligonucleotide vehicle containing a furan-side psoralen monoadduct to a 56 base long complementary oligonucleotide target strand and formed a specific cross-link at the target site with 365-nm UV. Subsequent BCR released the oligonucleotide vehicle and deposited the psoralen at the target site.     

Prediction of RNA secondary structure by maximizing pseudo-expected accuracy

Background  

Recent studies have revealed the importance of considering the entire distribution of possible secondary structures in RNA secondary structure predictions; therefore, a new type of estimator is proposed including the maximum expected accuracy (MEA) estimator. The MEA-based estimators have been designed to maximize the expected accuracy of the base-pairs and have achieved the highest level of accuracy. Those methods, however, do not give the single best prediction of the structure, but employ parameters to control the trade-off between the sensitivity and the positive predictive value (PPV). It is unclear what parameter value we should use, and even the well-trained default parameter value does not, in general, give the best result in popular accuracy measures to each RNA sequence.     

Prediction of RNA secondary structure by free energy minimization

RNA secondary structure is often predicted from sequence by free energy minimization. Over the past two years, advances have been made in the estimation of folding free energy change, the mapping of secondary structure and the implementation of computer programs for structure prediction. The trends in computer program development are: efficient use of experimental mapping of structures to constrain structure prediction; use of statistical mechanics to improve the fidelity of structure prediction; inclusion of pseudoknots in secondary structure prediction; and use of two or more homologous sequences to find a common structure.     

Vienna RNA secondary structure server

The Vienna RNA secondary structure server provides a web interface to the most frequently used functions of the Vienna RNA software package for the analysis of RNA secondary structures. It currently offers prediction of secondary structure from a single sequence, prediction of the consensus secondary structure for a set of aligned sequences and the design of sequences that will fold into a predefined structure. All three services can be accessed via the Vienna RNA web server at http://rna.tbi.univie.ac.at/.     

An RNA secondary structure workbench

A multiple approach to the study of RNA secondary structure is described which provides for the independent drawing of structures using base-pairing lists, for the generation of local structures in the form of hairpins, and for the generation of global structures by both Monte Carlo and dynamic programming methodologies. User-adjustable parameters provide for limiting the size of hairpin loops, bulges and inner loops, and constraints can be imposed relative to position-dependent base pairing.     

Structure of E. coli 16S RNA elucidated by psoralen crosslinking

E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T1 RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/- 15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes.     

Probing Tat peptide-TAR RNA interactions by psoralen photo-cross-linking

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. We have used a site-specific cross-linking method based on psoralen photochemistry to determine the effect of core residues from the Tat sequence on the protein orientation in the Tat-TAR complex and on the specificity of Tat-TAR binding. We synthesized two Tat fragments, Tat(42-72) and Tat(37-72), and incorporated a psoralen-modified amino acid at position 41 during solid-phase assembly of the peptides. We used these psoralen-Tat conjugates to form specific complexes with TAR RNA. Upon near-ultraviolet irradiation (360 nm), psoralen-Asp41-Tat(37-72) cross-linked to a single site in the TAR RNA sequence. The RNA-protein complex was purified and the cross-link site on TAR RNA was determined by primer extension analysis, which revealed that Asp41 of Tat is close to U42 of the lower stem region of TAR RNA. Specificity of the RNA-peptide cross-linking reactions was determined by competition experiments. Our results show that the addition of only four residues (Cys37-Thr40) from the Tat core region significantly enhanced the specificity of the Tat peptide-TAR interactions without altering the site or chemical nature of the cross-link. These studies provide new insights into RNA-protein recognition that could be useful in designing peptidomimetics for RNA targeting. Such psoralen-peptide conjugates provide a new class of probes for sequence-specific protein-nucleic acid interactions and could be used to selectively control gene expression or to induce site-directed mutations.     

Prediction of RNA secondary structure, including pseudoknotting, by computer simulation.

A computer program is presented which determines the secondary structure of linear RNA molecules by simulating a hypothetical process of folding. This process implies the concept of 'nucleation centres', regions in RNA which locally trigger the folding. During the simulation, the RNA is allowed to fold into pseudoknotted structures, unlike all other programs predicting RNA secondary structure. The simulation uses published, experimentally determined free energy values for nearest neighbour base pair stackings and loop regions, except for new extrapolated values for loops larger than seven nucleotides. The free energy value for a loop arising from pseudoknot formation is set to a single, estimated value of 4.2 kcal/mole. Especially in the case of long RNA sequences, our program appears superior to other secondary structure predicting programs described so far, as tests on tRNAs, the LSU intron of Tetrahymena thermophila and a number of plant viral RNAs show. In addition, pseudoknotted structures are often predicted successfully. The program is written in mainframe APL and is adapted to run on IBM compatible PCs, Atari ST and Macintosh personal computers. On an 8 MHz 8088 standard PC without coprocessor, using STSC APL, it folds a sequence of 700 nucleotides in one and a half hour.     

Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.     

General combinatorics of RNA secondary structure

The total number of RNA secondary structures of a given length with minimal hairpin loop length m(m>0) and with minimal stack length l(l>0) is computed, under the assumption that all base pairs can occur. Asymptotics are derived from the determination of recurrence relations of decomposition properties.