Natural DNA Sequences Research Articles

An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy

BackgroundWidely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired.ResultsThis paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15 bp homology was sufficient to initiate recombination, while 25 bp homology had the highest cloning efficiency. Inserts up to 4 kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP.ConclusionsABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.

The role of protein sequence and global conformation in DNA binding specificity of members of the Cro family

The Cro family of bacteriophage DNA‐binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are α‐helical and mixed α‐helical/β‐sheet, respectively, despite sharing 40% sequence identity. Both proteins are transcription factors that use a helix‐turn‐helix (HTH) motif as their primary DNA recognition moiety. However, the natural consensus DNA sequences of the proteins are different at three positions in each seven base‐pair half site. Fluorescence anisotropy measurements showed that wild‐type (WT) Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (KD) of 230 nM and 56 nM, respectively. WT Pfl 6 bound its noncognate site with KD = 1.99 μM and WT Xfaso 1 did not bind its noncognate site at all. To explore whether this change in binding specificity is due to differences in sequence in the HTH region or to global differences in their tertiary structures, mutations were introduced into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. The binding affinities of these variant proteins against the consensus binding sites were tested and preliminary results suggest that mutation of the Xfaso 1 sequence toward Pfl 6 allows it to bind the Pfl 6 consensus site. Funded by NSF (MCB‐0647390) and HHMI (52005889).

Sequence Context Effect on Strand Slippage in Natural DNA Primer–Templates

Strand slippage has been found to occur in primer-templates containing a templating thymine, cytosine, and guanine, leading to the formation of misaligned structures with a single-nucleotide bulge. If remained in the active site of low-fidelity polymerases during DNA replication, these misaligned structures can ultimately bring about deletion mutations. In this study, we performed NMR investigations on primer-template models containing a templating adenine. Similar to our previous results on guanine, adenine templates are also less prone to strand slippage than pyrimidine templates. Misalignment occurs only in primer-templates that form a terminal C·G or G·C base pair. Together with our previous findings on thymine, cytosine, and guanine templates, the present study reveals strand slippage can occur in any kind of natural templating bases during DNA replication, providing insights into the origin of mutation hotspots in natural DNA sequences. In addition to the type of incoming base upon misincorporation, the propensity of strand slippage in primer-templates depends also on the type of templating base, its upstream and downstream bases.

Synthesis and photophysical evaluation of a pyridinium 4-amino-1,8-naphthalimide derivative that upon intercalation displays preference for AT-rich double-stranded DNA

The synthesis, characterisation and solid state crystal structure of a cationic 4-amino-1,8-naphthalimide derivative (1) are described. The photophysical properties of 1 are shown to vary with the solvent polarity and H-bonding ability. The fluorescence of 1 is enhanced and blue-shifted in its 1:1 complex with 5'-adenosine-monophosphate while it is partially quenched and red-shifted in its complex with 5'-guanosine-monophosphate. Linear and circular dichroism measurements show that 1 binds to double-stranded DNA by intercalation. Comparative UV-visible and fluorescence studies with double stranded synthetic polynucleotides poly(dA-dT)(2) and poly(dG-dC)(2) show that 1 binds much more strongly to the AT polymer; 1 also has a strong preference for A-T rich sequences in natural DNA. Thermal denaturation measurements also reveal a much greater stabilisation of the double-stranded poly(dA-dT)(2) than of natural DNA.

A renormalization approach to describe charge transport in quasiperiodic dangling backbone ladder (DBL)-DNA molecules

We study the charge transport properties of a dangling backbone ladder (DBL)-DNA molecule focusing on a quasiperiodic arrangement of its constituent nucleotides forming a Rudin–Shapiro (RS) and Fibonacci (FB) Poly (CG) sequences, as well as a natural DNA sequence (Ch22) for the sake of comparison. Making use of a one-step renormalization process, the DBL-DNA molecule is modeled in terms of a one-dimensional tight-binding Hamiltonian to investigate its transmissivity and current–voltage (I–V) profiles. Beyond the semiconductor I–V characteristics, a striking similarity between the electronic transport properties of the RS quasiperiodic structure and the natural DNA sequence was found.

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009). Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

Zinc Finger Recombinases with Adaptable DNA Sequence Specificity

Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural ‘pseudosites’ are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene), mediated by zinc finger recombinases (ZFRs), chimaeric enzymes with linked zinc finger (DNA recognition) and recombinase (catalytic) domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences.

The Rate and Tract Length of Gene Conversion between Duplicated Genes

Interlocus gene conversion occurs such that a certain length of DNA fragment is non-reciprocally transferred (copied and pasted) between paralogous regions. To understand the rate and tract length of gene conversion, there are two major approaches. One is based on mutation-accumulation experiments, and the other uses natural DNA sequence variation. In this review, we overview the two major approaches and discuss their advantages and disadvantages. In addition, to demonstrate the importance of statistical analysis of empirical and evolutionary data for estimating tract length, we apply a maximum likelihood method to several data sets.

Monitoring DNA Binding to Escherichia coli Lactose Repressor Using Quartz Crystal Microbalance with Dissipation

Lactose repressor protein (LacI) functions as a negative transcription regulator in Escherichia coli by binding to the operator DNA sequence. Our understanding of the immobilized LacI function and the effect of ligand binding on the conformation of LacI-DNA complexes remains poorly understood. Here, we have examined the difference in functionality of wild-type and mutant LacI binding to the target DNA using quartz crystal microbalance with dissipation (QCM-D). To direct the orientation of LacI binding to the gold surface, residue 334 was substituted with cysteine (T334C) to generate a sulfur-gold linkage. Position 334 is located on the surface opposite the DNA-binding domain and remote from the site for inducer binding. With T334C immobilized on the gold surface, our sensors successfully detect operator binding as well as the release of the operator DNA from the repressor in the presence of inducer isopropyl-β-D-thiogalactoside (IPTG). Besides the natural operator DNA sequence (O(1)), a symmetric high-affinity DNA sequence (O(sym)), and a non-specific DNA (O(ns)) sequence with low affinity were also used. In addition, the impact of anti-inducer o-nitrophenyl-beta-d-fucoside (ONPF), which stabilizes LacI operator binding, was examined. The results from immobilized mutant LacI are in good agreement with known solution parameters for LacI-ligand interactions, demonstrating that QCM-D provides a rapid and efficient measurement of DNA binding and impact of ligands upon binding for this complex oligomeric protein.

The Modified DNA Identification Classification on Fuzzy Relation

We proposed a categorized method of DNA sequences matrix by FCM (fuzzy cluster means). FCM avoided the errors caused by the reduction of dimensions. It further reached comprehensive machine learning. In our experiment, there are 40 training data which are artificial samples, and we verify the proposed method with 182 natural DNA sequences. The result showed the proposed method enhanced the accuracy of the classification of genes from 76% to 93%.

A new alignment-free sequence analysis based on the distribution of <I>K</I>-tuple

Based on the distribution of K-tuple in complete genome, a method without doing sequence alignment to infer difference of biological sequence is proposed in this paper. The method can be used to measure the difference of distribution on K-tuple between the native DNA sequences and the corresponding randomized ones. Applied to construct phylogenetic trees of the complete mitochondrial genomes of 26 species of placental mammals, with K increasing, it yields phylogenetic trees of which the classification effect increasingly matches the result widely recognised by the biological field. The final results show that the phylogenetic trees built by this method is more reasonable than by other alignment-free sequence methods.

Archaeal Intrinsic Transcription Termination In Vivo

Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a beta-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5'-TTTTTTTT (T(8)) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t(mcrA)) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression approximately 90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region. Template DNAs were amplified from each T. kodakarensis strain, and transcription in vitro by T. kodakarensis RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5'-AAAAAAAA (A(8)) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination.

Protein-binding microarrays: probing disease markers at the interface of proteomics and genomics

DNA-binding proteins mediate a variety of crucial molecular functions, such as transcriptional regulation and chromosome maintenance, replication and repair, which in turn control cell division and differentiation. The roles of these proteins in disease are currently being investigated using microarray-based approaches. However, these assays can be difficult to adapt to routine diagnosis of complex diseases such as cancer. Here, we review promising alternative approaches involving protein-binding microarrays (PBMs) that probe the interaction of proteins from crude cell or tissue extracts with large collections of synthetic or natural DNA sequences. Recent studies have demonstrated the use of these novel PBM approaches to provide rapid and unbiased characterization of DNA-binding proteins as molecular markers of disease, for example cancer progression or infectious diseases.

Efficient Replication Bypass of Size‐Expanded DNA Base Pairs in Bacterial Cells

Supersize me! Size-expanded DNA bases (xDNA) are able to encode natural DNA sequences in replication. In vitro experiments with a DNA polymerase show nucleotide incorporation opposite the xDNA bases with correct pairing. In vivo experiments using E. coli show that two xDNA bases (xA and xC, see picture) encode the correct replication partners.

A Direct and Nondestructive Approach To Determine the Folding Structure of the I-Motif DNA Secondary Structure by NMR

I-motifs are four-stranded DNA secondary structures formed in C-rich DNA sequences and consist of parallel-stranded DNA duplexes zipped together in an antiparallel orientation by intercalated, hemiprotonated cytosine(+)-cytosine base pairs. I-motif structures have been indicated to form in various regions of the human genome as well as in nanotechnological applications. While NMR is a major tool for structural studies of I-motifs, the determination of the folding topologies of unimolecular I-motifs has been a challenging and arduous task using conventional NMR spectral assignment strategies, due to the inherent sequence redundancy of the C-rich strands in the formation of unimolecular I-motif structures. We report here a direct and nondestructive method that can be utilized to unambiguously determine the hemiprotonated C(+)-C base pairs and thus the folding topology of unimolecular I-motif structures formed from native C-rich DNA sequences. The reported approach uses affordable low-enrichment site-specific labeling. More significantly, the reported method can directly and unambiguously determine the equilibrating multiple conformations coexisting in a single DNA sequence, which would be a very difficult task using conventional assignment strategies. Additionally, this method can be applied to the direct detection of the base-paired thymines that are involved in the capping structures.

Investigation of transcription factor Ndt80 affinity differences for wild type and mutant DNA: A molecular dynamics study

Molecular dynamics simulations and free energy calculations have been performed on the transcription factor Ndt80 either in complex with the native DNA sequence or with a mutant DNA with a switched central base pair, C5-G5' to G5-C5'. This mutant has been shown to have a 100-fold decrease in binding affinity of Ndt80, and in this study we explain this both structurally and energetically. The major interactions between the protein and the DNA were maintained in the simulations, apart from around the mutation site. The crystal structure of the Ndt80-DNA complex revealed that R177 makes a base specific bidentate interaction with G5' which is part of a conserved 5'-YpG-3' step. In the simulation with the mutant DNA, the side chain of R177 changes conformation and makes three new stable hydrogen bonds to the DNA backbone. This in turn induces a conformational change in the DNA backbone of the T6'-G5' step from the unusual BII state to the canonical BI state. The affinity difference for the protein-DNA complex with the native DNA compared with the mutant DNA is only about 3 kcal/mol. The free energy calculations of the base pair switch indicated a larger difference than what was found experimentally, about 7.7 kcal/mol, but this is explained in structural terms using the 10 ns simulations of the solvated complexes and the rearrangement of the R177 side chain.

DNA Sequences in gal Operon Override Transcription Elongation Blocks

The DNA loop that represses transcription from galactose (gal) promoters is infrequently formed in stationary-phase cells because the concentration of the loop architectural protein HU is significantly low at that state, resulting in expression of the operon in the absence of the gal inducer d-galactose. Unexpectedly, transcription from the gal promoters under these conditions overrides physical block because of the presence of the Gal repressor bound to an internal operator (OI) located downstream of the promoters. We have shown here that although a stretch of pyrimidine residues (UUCU) in the RNA:DNA hybrid located immediately upstream of OI weakens the RNA:DNA hybrid and favors RNA polymerase (RNAP) pausing and backtracking, a stretch of purines (GAGAG) in the RNA present immediately upstream of the pause sequence in the hybrid acts as an antipause element by stabilizing the RNA:DNA duplex and preventing backtracking. This facilitates forward translocation of RNAP, including overriding of the DNA-bound Gal repressor barrier at OI. When the GAGAG sequence is separated from the pyrimidine sequence by a 5-bp DNA insertion, RNAP backtracking is favored from a weak hybrid to a more stable hybrid. RNAP backtracking is sensitive to Gre factors, d-galactose, and antisense oligonucleotides. The ability of a native DNA sequence to override transcription elongation blocks in the gal operon uncovers a previously unknown way of regulating gal metabolism in Escherichia coli. It also explains the synthesis of gal enzymes in the absence of inducer for biosynthetic reactions.

Spectrum-Based De Novo Repeat Detection in Genomic Sequences

A novel approach to the detection of genomic repeats is presented in this paper. The technique, dubbed SAGRI (Spectrum Assisted Genomic Repeat Identifier), is based on the spectrum (set of sequence k-mers, for some k) of the genomic sequence. Specifically, the genome is scanned twice. The first scan (FindHit) detects candidate pairs of repeat-segments, by effectively reconstructing portions of the Euler path of the (k-1)-mer graph of the genome only in correspondence with likely repeat sites. This process produces candidate repeat pairs, for which the location of the leftmost term is unknown. Candidate pairs are then subjected to validation in a second scan, in which the genome is labelled for hits in the (much smaller) spectrum of the repeat candidates: high hit density is taken as evidence of the location of the first segment of a repeat, and the pair of segments is then certified by pairwise alignment. The design parameters of the technique are selected on the basis of a careful probabilistic analysis (based on random sequences). SAGRI is compared with three leading repeat-finding tools on both synthetic and natural DNA sequences, and found to be uniformly superior in versatility (ability to detect repeats of different lengths) and accuracy (the central goal of repeat finding), while being quite competitive in speed. An executable program can be downloaded at http://sagri.comp.nus.edu.sg.

G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.