Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.     

The computer simulation of RNA folding involving pseudoknot formation.

The algorithm and the program for the prediction of RNA secondary structure with pseudoknot formation have been proposed. The algorithm simulates stepwise folding by generating random structures using Monte Carlo method, followed by the selection of helices to final structure on the basis of both their probabilities of occurrence in a random structure and free energy parameters. The program versions have been tested on ribosomal RNA structures and on RNAs with pseudoknots evidenced by experimental data. It is shown that the simulation of folding during RNA synthesis improves the results. The introduction of pseudoknot formation permits to predict the pseudoknotted structures and to improve the prediction of long-range interactions. The computer program is rather fast and allows to predict the structures for long RNAs without using large memory volumes in usual personal computer.     

How RNA folds.

We describe the RNA folding problem and contrast it with the much more difficult protein folding problem. RNA has four similar monomer units, whereas proteins have 20 very different residues. The folding of RNA is hierarchical in that secondary structure is much more stable than tertiary folding. In RNA the two levels of folding (secondary and tertiary) can be experimentally separated by the presence or absence of Mg2+. Secondary structure can be predicted successfully from experimental thermodynamic data on secondary structure elements: helices, loops, and bulges. Tertiary interactions can then be added without much distortion of the secondary structure. These observations suggest a folding algorithm to predict the structure of an RNA from its sequence. However, to solve the RNA folding problem one needs thermodynamic data on tertiary structure interactions, and identification and characterization of metal-ion binding sites. These data, together with force versus extension measurements on single RNA molecules, should provide the information necessary to test and refine the proposed algorithm.     

A dynamic programming algorithm for finding alternative RNA secondary structures.

Dynamic programming algorithms that predict RNA secondary structure by minimizing the free energy have had one important limitation. They were able to predict only one optimal structure. Given the uncertainties of the thermodynamic data and the effects of proteins and other environmental factors on structure, the optimal structure predicted by these methods may not have biological significance. We present a dynamic programming algorithm that can determine optimal and suboptimal secondary structures for an RNA. The power and utility of the method is demonstrated in the folding of the intervening sequence of the rRNA of Tetrahymena. By first identifying the major secondary structures corresponding to the lowest free energy minima, a secondary structure of possible biological significance is derived.     

RNA-SSPT: RNA Secondary Structure Prediction Tools

The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.     

An implementation of the Gillespie algorithm for RNA kinetics with logarithmic time update

In this paper I outline a fast method called KFOLD for implementing the Gillepie algorithm to stochastically sample the folding kinetics of an RNA molecule at single base-pair resolution. In the same fashion as the KINFOLD algorithm, which also uses the Gillespie algorithm to predict folding kinetics, KFOLD stochastically chooses a new RNA secondary structure state that is accessible from the current state by a single base-pair addition/deletion following the Gillespie procedure. However, unlike KINFOLD, the KFOLD algorithm utilizes the fact that many of the base-pair addition/deletion reactions and their corresponding rates do not change between each step in the algorithm. This allows KFOLD to achieve a substantial speed-up in the time required to compute a prediction of the folding pathway and, for a fixed number of base-pair moves, performs logarithmically with sequence size. This increase in speed opens up the possibility of studying the kinetics of much longer RNA sequences at single base-pair resolution while also allowing for the RNA folding statistics of smaller RNA sequences to be computed much more quickly.     

Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.     

The single strand conformational analysis of cattle and human single nucleotide polymorphisms may be biased towards specific sequence motifs that minimize local secondary structure of single strand DNA

Single strand conformational polymorphisms (SSCP) resulting from point mutations were found to be associated preferentially with two DNA sequence motifs. These motifs are (1) three or more of the same base but in which the polymorphism is not due to length variation and (2) a region of polypurine or polypyrimidine bases. These motifs were identified after SSCP alleles from cattle were sequenced. The sequence difference and flanking sequence for each single nucleotide polymorphism are shown. The motifs were also found in SSCP from humans chosen at random from the literature, in which the alleles had been sequenced. There is a low level of complementarity of adjacent bases in these motifs and they should represent regions of low secondary structure in the single stranded DNA. Regions of high secondary structure, such as palindromes, were found in the same sample to have allelic variation that was not detected by SSC analysis. These results give a rule of thumb for selecting the particular part of a DNA fragment to be selected for testing for polymorphisms, but this rule clashes with rules used to design primers to amplify sequences using the PCR, namely, minimise hydrogen bonding within and between primers and reduce self-complementarity.     

Consensus folding of unaligned RNA sequences revisited.

As one of the earliest problems in computational biology, RNA secondary structure prediction (sometimes referred to as "RNA folding") problem has attracted attention again, thanks to the recent discoveries of many novel non-coding RNA molecules. The two common approaches to this problem are de novo prediction of RNA secondary structure based on energy minimization and the consensus folding approach (computing the common secondary structure for a set of unaligned RNA sequences). Consensus folding algorithms work well when the correct seed alignment is part of the input to the problem. However, seed alignment itself is a challenging problem for diverged RNA families. In this paper, we propose a novel framework to predict the common secondary structure for unaligned RNA sequences. By matching putative stacks in RNA sequences, we make use of both primary sequence information and thermodynamic stability for prediction at the same time. We show that our method can predict the correct common RNA secondary structures even when we are given only a limited number of unaligned RNA sequences, and it outperforms current algorithms in sensitivity and accuracy.     

Transverse formamide gradients as a simple and easy way to optimise DNA single-strand conformation polymorphism analysis.

Although widely used, the detection of DNA mutations by the single-strand conformation polymorphism (SSCP) method is often hampered by the need to examine a large set of electrophoretic conditions in order to select the one suited to the DNA sequence under study. We show here that the use of transverse chemical gradient gels allows for a quick and easy optimisation of SSCP analysis, as exemplified on two mutations in exon 2 of the alpha-1-antitrypsin gene.     

Prediction of common folding structures of homologous RNAs.

We have developed an algorithm and a computer program for simultaneously folding homologous RNA sequences. Given an alignment of M homologous sequences of length N, the program performs phylogenetic comparative analysis and predicts a common secondary structure conserved in the sequences. When the structure is not uniquely determined, it infers multiple structures which appear most plausible. This method is superior to energy minimization methods in the sense that it is not sensitive to point mutation of a sequence. It is also superior to usual phylogenetic comparative methods in that it does not require manual scrutiny for covariation or secondary structures. The most plausible 1-5 structures are produced in O(MN2 + N3) time and O(N2) space, which are the same requirements as those of widely used dynamic programs based on energy minimization for folding a single sequence. This is the first algorithm probably practical both in terms of time and space for finding secondary structures of homologous RNA sequences. The algorithm has been implemented in C on a Sun SparcStation, and has been verified by testing on tRNAs, 5S rRNAs, 16S rRNAs, TAR RNAs of human immunodeficiency virus type 1 (HIV-1), and RRE RNAs of HIV-1. We have also applied the program to cis-acting packaging sequences of HIV-1, for which no generally accepted structures yet exist, and propose potentially stable structures. Simulation of the program with random sequences with the same base composition and the same degree of similarity as the above sequences shows that structures common to homologous sequences are very unlikely to occur by chance in random sequences.     

Probing of DNA structure with osmium tetroxide,2,2''-bipyridine. Adduct-specific antibodies.

Antibodies against DNA modified with a single-strand selective probe, OsO4 in complex with 2,2'-bipyridine (Os,bipy), were raised in rabbits. These antibodies were fractionated using affinity column chromatography and fractions S89-II and S89-III characterized as highly specific for DNA-Os,bipy adduct with no cross reactivity to at least 1000-fold excess of unmodified DNA, RNA and Os,bipy-modified and unmodified proteins. Cross-reactivity to Os,bipy-modified RNA was very small. S89-II showed no cross-reactivity to DNA modified with OsO4 complexed with tetramethylethylenediamine or with bathophenanthroline disulphonic acid and to DNA oxidized with KMnO4. It cross-reacted, however, with DNA modified with OsO4,1,10-phenanthroline complex. The limit of detection of immunodot-blot analysis of extensively Os,bipy-modified DNA was below 0.5 pg. Small extent of Os,bipy-modification of supercoiled and linearized plasmids can be detected by DNA gel retardation and immunoblotting techniques. E. coli cells contain DNA regions in which bases are accessible to the single-strand selective probe.     

The use of counter-current distribution to examine the effect of damaging agents on DNA

Mammalian DNA's were separated using a counter-current distribution system for demonstrating alteration in secondary structure after heat denaturation and drug treatment. By using this method a complete separation of native and denatured DNA was achieved. Although the separation of DNA depends on the temperature used for denaturation, the counter-current distribution pattern did not follow exactly the hyperchromic shift. The results suggest that counter-current distribution offers a complementary approach for the study of DNA secondary structure as this method reveals alterations occurring over a wider temperature range than the increase in ultraviolet absorption. The changes in distribution pattern demonstrate cross-linkage occurring with nitrogen mustard and single-strand breaks following methylene dimethanesulphonate (MDMS) treatment in vitro.     

Prediction of RNA pseudoknots using heuristic modeling with mapping and sequential folding

Predicting RNA secondary structure is often the first step to determining the structure of RNA. Prediction approaches have historically avoided searching for pseudoknots because of the extreme combinatorial and time complexity of the problem. Yet neglecting pseudoknots limits the utility of such approaches. Here, an algorithm utilizing structure mapping and thermodynamics is introduced for RNA pseudoknot prediction that finds the minimum free energy and identifies information about the flexibility of the RNA. The heuristic approach takes advantage of the 5' to 3' folding direction of many biological RNA molecules and is consistent with the hierarchical folding hypothesis and the contact order model. Mapping methods are used to build and analyze the folded structure for pseudoknots and to add important 3D structural considerations. The program can predict some well known pseudoknot structures correctly. The results of this study suggest that many functional RNA sequences are optimized for proper folding. They also suggest directions we can proceed in the future to achieve even better results.     

Practicality and time complexity of a sparsified RNA folding algorithm

Commonly used RNA folding programs compute the minimum free energy structure of a sequence under the pseudoknot exclusion constraint. They are based on Zuker's algorithm which runs in time O(n(3)). Recently, it has been claimed that RNA folding can be achieved in average time O(n(2)) using a sparsification technique. A proof of quadratic time complexity was based on the assumption that computational RNA folding obeys the "polymer-zeta property". Several variants of sparse RNA folding algorithms were later developed. Here, we present our own version, which is readily applicable to existing RNA folding programs, as it is extremely simple and does not require any new data structure. We applied it to the widely used Vienna RNAfold program, to create sibRNAfold, the first public sparsified version of a standard RNA folding program. To gain a better understanding of the time complexity of sparsified RNA folding in general, we carried out a thorough run time analysis with synthetic random sequences, both in the context of energy minimization and base pairing maximization. Contrary to previous claims, the asymptotic time complexity of a sparsified RNA folding algorithm using standard energy parameters remains O(n(3)) under a wide variety of conditions. Consistent with our run-time analysis, we found that RNA folding does not obey the "polymer-zeta property" as claimed previously. Yet, a basic version of a sparsified RNA folding algorithm provides 15- to 50-fold speed gain. Surprisingly, the same sparsification technique has a different effect when applied to base pairing optimization. There, its asymptotic running time complexity appears to be either quadratic or cubic depending on the base composition. The code used in this work is available at: .     

The nucleotide sequence of threonine transfer RNA coded by bacteriophage T4.

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.     

Detection of point mutations in DNA using capillary electrophoresis in a polymer network

The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.     

Simulating the Folding Pathway of RNA Secondary Structure Using the Modified Ant Colony Algorithm

<正> A new method for simulating the folding pathway of RNA secondary structure using the modified ant colony algorithmis proposed.For a given RNA sequence,the set of all possible stems is obtained and the energy of each stem iscalculated and stored at the initial stage.Furthermore,a more realistic formula is used to compute the energy ofmulti-branch loop in the following iteration.Then a folding pathway is simulated,including such processes as constructionof the heuristic information,the rule of initializing the pheromone,the mechanism of choosing the initial andnext stem and the strategy of updating the pheromone between two different stems.Finally by testing RNA sequences withknown secondary structures from the public databases,we analyze the experimental data to select appropriate values forparameters.The measure indexes show that our procedure is more consistent with phylogenetically proven structures thansoftware RNAstructure sometimes and more effective than the standard Genetic Algorithm.