Random DNA Sequence Research Articles

Viral Reverse Transcriptases Show Selective High Affinity Binding to DNA-DNA Primer-Templates that Resemble the Polypurine Tract

Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment)-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT) showed that primers mimicking the 3′ end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5′-AAAAGAAAAGGGGGG-3′) were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV)) and one retrotransposon (Ty3) RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3′ end (AMV PPT: 5′-AGGGAGGGGGA-3′; MuLV PPT: 5′-AGAAAAAGGGGGG-3′). In contrast, Ty3, whose PPT lacks a G tract (5′-GAGAGAGAGGAA-3′) showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs) mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3′ end of the PPT where extension can occur.

ALAE

We study the problem of local alignment, which is finding pairs of similar subsequences with gaps. The problem exists in biosequence databases. BLAST is a typical software for finding local alignment based on heuristic, but could miss results. Using the Smith-Waterman algorithm, we can find all local alignments in O( mn ) time, where m and n are lengths of a query and a text, respectively. A recent exact approach BWT-SW improves the complexity of the Smith-Waterman algorithm under constraints, but still much slower than BLAST. This paper takes on the challenge of designing an accurate and efficient algorithm for evaluating local-alignment searches, especially for long queries. In this paper, we propose an efficient software called ALAE to speed up BWT-SW using a compressed suffix array. ALAE utilizes a family of filtering techniques to prune meaningless calculations and an algorithm for reusing score calculations. We also give a mathematical analysis and show that the upper bound of the total number of calculated entries using ALAE could vary from 4.50 mn 0.520 to 9.05 mn 0.896 for random DNA sequences and vary from 8.28 mn 0.364 to 7.49 mn 0.723 for random protein sequences. We demonstrate the significant performance improvement of ALAE on BWT-SW using a thorough experimental study on real biosequences. ALAE guarantees correctness and accelerates BLAST for most of parameters.

Cell-Penetrating RNAs: New Keys to the Castle

The plasma membrane represents the walls of the cellular fortress. It maintains cellular integrity by separating the insides from the outside, and the gatekeepers within these walls are very selective about which molecules are allowed in or kept out. Since the pioneering work on bacterial transformation by Griffith, Avery, and others more than half a century ago,1,2 a series of technologies has been developed to deliver payloads across membranes. In a forthcoming article in Molecular Therapy, Magalhaes et al. describe a new way to unlock one of the fortress gates in the form of aptamers they dub “c1” and “Otter.”3 These aptamers bind and penetrate into a diverse collection of mouse and human cells, including primary cells. Although there are many unanswered questions about the biology of these aptamers, it seems plausible that they could prove useful as tools for delivering molecular payloads for basic research and therapeutic applications.

Liquid crystal self-assembly of random-sequence DNA oligomers

In biological systems and nanoscale assemblies, the self-association of DNA is typically studied and applied in the context of the evolved or directed design of base sequences that give complementary pairing, duplex formation, and specific structural motifs. Here we consider the collective behavior of DNA solutions in the distinctly different regime where DNA base sequences are chosen at random or with varying degrees of randomness. We show that in solutions of completely random sequences, corresponding to a remarkably large number of different molecules, e.g., approximately 10(12) for random 20-mers, complementary still emerges and, for a narrow range of oligomer lengths, produces a subtle hierarchical sequence of structured self-assembly and organization into liquid crystal (LC) phases. This ordering follows from the kinetic arrest of oligomer association into long-lived partially paired double helices, followed by reversible association of these pairs into linear aggregates that in turn condense into LC domains.

Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.

Target site cleavage by the monomeric restriction enzyme BcnI requires translocation to a random DNA sequence and a switch in enzyme orientation

Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5′-CCSGG-3′ (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.

5‐Fluorouracil‐based chemotherapy for colorectal cancer and MTHFR/MTRR genotypes

5‐Fluorouracil‐based chemotherapy for colorectal cancer and <i>MTHFR</i>/<i>MTRR</i> genotypes

Force induced unzipping of DNA with long range correlated noise

We derive and solve a Fokker–Planck equation for the stationary distribution of the freeenergy, in a model of unzipping of double-stranded DNA under external force.The autocorrelation function of the random DNA sequence can be of a generalform, including long range correlations. In the case of Ornstein–Uhlenbeck noise,characterized by a finite correlation length, our result reduces to the exact result ofAllahverdyan et al, with the average number of unzipped base pairs going as⟨X⟩ ∼ 1/f2 in the white noiselimit, where f is the deviation from the critical force. In the case of long range correlatednoise, where the integrated autocorrelation is divergent, we find that⟨X⟩ is finiteat f = 0, with its value decreasing as the correlations become of longer range. This shows that longrange correlations actually stabilize the DNA sequence against unzipping. Our result is alsoin agreement with the findings of Allahverdyan et al obtained using numerical generation ofthe long range correlated noise.

Single-Molecule Studies of Human RNA Polymerase Ii Transcription Initiation: Core-Promoter Recognition

We have developed a robust experimental platform that allows us to study the assembly dynamics of complex, multi-components biochemical systems at the single-molecule level. A key element of our implementation was the refinement of surface passivation protocols for single-molecule experiments, to achieve a 10- to 30-fold reduction in non-specific interactions for the various components of the Human RNA Polymerase II machinery. The reduced background of this optimized system allowed us to monitor the assembly of fluorescent transcription machinery components on promoter-containing DNA molecules, using the mutli-color (sub-)nanometer co-localization capabilities of our recently developed actively-stabilized microscope setup (Pertsinidis et al., Nature 2010).In addition, we applied single-molecule spectroscopy tools, and as well as numerical modeling, to start dissecting the cascade of conformational rearrangements during Pre-initiation Complex (PIC) formation. Comprised of the General Transcription Factors (GTFs) TFIID/A/B/F/E/H and RNAPol II, assembly of this g50-polypeptide, Mega-Dalton complex commences with recognition of core-promoter elements by the TFIID complex. We observe that the TATA-binding (TBP) sub-unit of TFIID binds the consensus AdMLP core promoter with a transition to the severely kinked/partially unwound TATA-box state. Interestingly, we observe that TBP can also recognize variant TATA elements, resulting in structures with intermediate degrees of DNA deformation. Finally, our observations reveal additional conformational changes upon TFIIA (and to some extent TFIIB) recruitment, suggesting a mechanism for regulating further progression of the PIC assembly on strong as well as weak TATA-containing promoters but not on random DNA sequences.

Functional Nucleic Acids as Molecular Recognition Elements for Small Organic and Biological Molecules

The abundance of small molecules in biological systems, the wide-spread usage of synthetic small molecules as drugs and biological probes, and the escalation of industrial pollutants in our environment, underscore the importance of detection of small molecules for many disciplines. Functional nucleic acids (FNAs) are single-stranded DNA or RNA sequences that are capable of carrying out ligand binding (aptamers), catalysis (nucleic acid enzymes) or both functions (aptazymes). Many FNAs have been shown to be suitable molecular recognition elements for small-molecule targets. In this article, we will present a focused review on FNAs for small molecule binding and detection. First, we will discuss the technique of “in vitro selection” by which artificial FNAs can be isolated from random-sequence DNA or RNA pools. This will be followed by a survey of aptamers for small molecules isolated to date. Next, the diverse functions of natural aptamers, as part of riboswitches (metabolite-sensing RNA regulatory systems that exist in many organisms) will be explored. Efforts in creating aptazymes will also be presented. Finally, we will examine numerous applications of aptamers and aptazymes in the development of fluorescent, colorimetric and electrochemical biosensors and discuss some emerging applications concerning FNAs.

A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis

Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve.

Characterization of non-8–17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.

The inherent occurrence of complex intron-rich spliceosomal split genes, including regulatory and splicing elements, within pre-biotic random genetic sequences

AbstractGrowing evidence indicates that complex intron-rich split genes and an advanced spliceosome existed in the earliest eukaryote, and possibly the first life form. We sought to examine how these split genes could have originated in the prebiotic system. We previously found that split coding sequences for complex proteins occur in abundance in random DNA sequences (P. Senapathy, et al, accompanying paper). This study demonstrates that a full complement of exons, introns and regulatory and splicing elements could have also occurred inherently within pre-biotic chemistry by chance. By comparing the characteristics of split genes found in computer-generated random genetic sequences with those of several extant eukaryotes, we show that an abundance of intron-rich split genes akin to those present in modern eukaryotes could have existed in the prebiotic system. These findings answer the post-genomic question of why the earliest life form contained highly complex intron-rich split genes, and, in conjunction with our companion study, show how they could encode a complex spliceosome.

Parallel genome assembly from pre-biotic split-genes: A solution for the mosaic genome conundrum

AbstractThe classical concept that all complex life evolved from a simple bacterium-like life form has been questioned by comparative genomics and proteomics. Several discoveries, including the occurrence of entirely new genes in unexpected life forms without any precursors, and the random mosaic distribution of genes across organisms in higher taxa undermine the principal assertions of this theory. We present a new model of the pre-biotic parallel evolution of eukaryotic genomes starting with the structurally complex split genes, which are far more probable of supporting biological information and of occurring in random DNA sequences than contiguous genes. By comparing the mosaic patterns of simulated genomes and extant genomes, we show that genomes arising from simulated assembly of random split genes form the same gene distribution patterns observed in extant eukaryotes. This parallel genome origin model eliminates the post-genomic and classical conundrums faced by the linear branching evolution model.

Rational Design, Synthesis, and DNA Binding Properties of Novel Sequence‐Selective Peptidyl Congeners of Ametantrone

Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti-neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10-anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well-defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC-rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4-bis-[Gly-(L-Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases.

Heterobifunctional modification of DNA for conjugation to solid surfaces

Many biosensors, DNA arrays, and next-generation DNA sequencing technologies need common methods for end modification of random DNA sequences generated from a sample of DNA. Surface immobilization of chemically modified DNA is often the first step in creating appropriate sensing platforms. We describe a simple technique for efficient heterobifunctional modification of arbitrary double-stranded DNA fragments with chosen chemical groups. The modification requires the use of short (10-20 base pairs) synthetic adaptors having desired terminal functional groups and installs known sequences, which can be used for hybridization of primers in the sequencing-by-synthesis approaches. The method, based on ligation under optimized conditions, is selective and provides high yields of the target heterobifunctional DNA product. An additional two-step procedure can be applied to select further for the desired bifunctionalized product using PCR amplification with a chemically modified primer. Both functional groups in the modified DNA are chemically active and can be used in surface immobilization of the DNA strands to create the surface of a biosensor or sequencing chip.

Long-range correlations and charge transport properties of DNA sequences

By using Hurst's analysis and transfer approach, the rescaled range functions and Hurst exponents of human chromosome 22 and enterobacteria phage lambda DNA sequences are investigated and the transmission coefficients, Landauer resistances and Lyapunov coefficients of finite segments based on above genomic DNA sequences are calculated. In a comparison with quasiperiodic and random artificial DNA sequences, we find that λ-DNA exhibits anticorrelation behavior characterized by a Hurst exponent 0.5 < H < 1 while, as far as the segments selected in our Letter are concerned, Ch22 sequence displays a transition from correlation behavior to anticorrelation behavior. The resonant peaks of the transmission coefficient in genomic sequences can survive in longer sequence length than in random sequences but in shorter sequence length than in quasiperiodic sequences. It is shown that the genomic sequences have long-range correlation properties to some extent but the correlations are not strong enough to maintain the scale invariance properties.

Optical, Redox, and DNA‐Binding Properties of Phenanthridinium Chromophores: Elucidating the Role of the Phenyl Substituent for Fluorescence Enhancement of Ethidium in the Presence of DNA

The phenanthridinium chromophores 5-ethyl-6-phenylphenanthridinium (1), 5-ethyl-6-methylphenanthridinium (2), 3,8-diamino-5-ethyl-6-methylphenanthridinium (3), and 3,8-diamino-5-ethyl-6-(4-N,N-diethylaminophenyl)phenanthridinium (4) were characterized by their optical and redox properties. All dyes were applied in titration experiments with a random-sequence 17mer DNA duplex and their binding affinities were determined. The results were compared to well-known ethidium bromide (E). In general, this set of data allows the influence of substituents in positions 3, 6, and 8 on the optical properties of E to be elucidated. Especially, compound 4 was used to compare the weak electron-donating character of the phenyl substituent at position 6 of E with the more electron-donating 4-N,N-diethylaminophenyl group. Analysis of all of the measurements revealed two pairs of chromophores. The first pair, consisting of 1 and 2, lacks the amino groups in positions 3 and 8, and, as a result, these dyes exhibit clearly altered optical and electrochemical properties compared with E. In the presence of DNA, a significant fluorescence quenching was observed. Their binding affinity to DNA is reduced by nearly one order of magnitude. The electronic effect of the phenyl group in position 6 on this type of dye is rather small. The properties of the second set, 3 and 4, are similar to E due to the presence of the two strongly electron-donating amino groups in positions 3 and 8. However, in contrast to 1 and 2, the electron-donating character of the substituent in position 6 of 3 and 4 is critical. The binding, as well as the fluorescence enhancement, is clearly related to the electron-donating effect of this substituent. Accordingly, compound 4 shows the strongest binding affinity and the strongest fluorescence enhancement. Quantum chemical calculations reveal a general mechanism related to the twisted intramolecular charge transfer (TICT) model. Accordingly, an increase of the twist angle between the phenyl ring in position 6 and the phenanthridinium core opens a nonradiative channel in the excited state that depends on the electron-donating character of the phenyl group. Access to this channel is hindered upon binding to DNA.

Genetic diversity of Tunisian accessions of Aegilops geniculata Roth and durum wheats (Triticum durum Desf.) using RAPD markers

Genetic diversity of three durum wheat cultivars (Triticum durum Desf.) and thirteen Aegilops geniculata Roth accessions belonging to different regions of Tunisia (in North and Central) was evaluated using random amplified polymorphic DNA (RAPD) markers. Nineteen arbitrary universal primers were used for the amplification of random DNA sequences. Data were analysed with the SIMQUAL program using the NTSYS-pc. Phylogenetic diagram was drowning using the UPGMA algorithm. Results indicate an important inter—specific polymorphism between Aegilops and durum wheat. Ae. geniculata accessions revealed a high level of polymorphism (71.27%) than wheat cultivars (39.76%). Two main groups were represented by cluster and principal component analyses. The first group is formed by Ae. geniculata accessions and the second is constituted only by durum wheat cultivars. Consequently, RAPD markers separate clearly Ae. geniculata accessions and durum wheat cultivars.

Design and assessment of binary DNA for nanopore sequencing

DNA sequencing using an array of nanometer-sized pores (nanopores) offer an exciting option for third-generation sequencing, which will allow faster and cheaper sequencing with minimal sample pre-processing. When a voltage is applied through a nanopore in a conducting fluid, a slight electric current is observed, the strength of which depends on the structure of the nanopore. When a DNA molecule passed through a nanopore, with an applied voltage, the current detected through the nanopore will differ for each base due to their differential effect on the structure of nanopore. However, current nanopore platforms cannot fully differentiate between single nucleotides due to their fast passage through the nanopore. To overcome this, Meller et al. proposed a strategy where the sequence detection is fluorescence based and involves passage of predesigned short oligonucleotides, so-called binary DNAs, encoding nucleotides [1]. In the current model, there are two binary DNA sequences and their binary combination encodes the DNA bases. Two molecular beacons complementary to the binary DNAs, each carrying a different fluorophore are also required for the fluorescence detection of DNA sequences. The genomic DNA is sheared and every 24 bases of sequence is converted to a DNA molecule consisting of corresponding binary DNAs encoding the matching bases. The binary-encoded DNA is then hybridised with molecular beacons. Once the binary-encoded DNA passes through the nanopore, the beacons are separated from the original strand and a short burst of light is emitted which is then used to determine the base information to sequence the DNA. We have designed sequences that serve as binary DNAs and corresponding molecular beacons. They have balanced GC content, minimal secondary structure, no cross-hybridisation and no occurrence in the human genome. A software pipeline either generates random DNA sequences or parses sequences from Archaea genomes. It then filters sequences by their GC content, repeat content, complexity, self hybridisation and occurrence in the human genome. Sequences that meet the desired criteria are then paired and tested further (e.g. cross-hybridisation). One set of a successful pair of binary DNA was assessed using the Biacore instrument, which allows interaction analysis in a label-free fashion. Our results confirmed that there is no cross hybridisation between the binary DNA sequences and they have similar hybridisation kinetics. We are currently testing the performance of molecular beacons and different hybridisation conditions (e.g. salt concentration, temperature). Our aim is to sequence an actual binary converted DNA using an array of nanopores.