Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.     

Nanomaterial-based electrochemical DNA sensing strategies

DNA sensing strategies have recently been varieted with the number of attempts at the development of different biosensor devices based on nanomaterials, which will further become DNA microchip systems. The investigations at the side of material science in connection with electrochemical biosensors open new directions for detection of specific gene sequences, and nucleic acid-ligand interactions.An overview is reported here about nanomaterial-based electrochemical DNA sensing strategies principally performed for the analysis of specific DNA sequences and the quantification of nucleic acids. Important features of electrochemical DNA sensing strategies, along with new developments based on nanomaterials are described and discussed.     

Self-quenching smart probes as a platform for the detection of sequence-specific UV-induced DNA photodamage

Molecular beacons (MBs) are sensitive probes for many DNA sequence-specific applications, such as DNA damage detection, but suffer from technical and cost limitations. We have designed smart probes with self-quenching properties as an alternative to molecular beacons to monitor sequence-specific UV-induced photodamage of oligonucleotides. These probes have similar stem-loop structural characteristics as molecular beacons, but quenching is achieved instead via photoinduced intramolecular electron transfer by neighboring guanosine residues. Our results indicate that the probes are sensitive enough to detect nanomolar target concentrations and are specific enough to discriminate single-base damage. When the probes were used to monitor UV-induced photodamage in oligonucleotide sequences that differ by a single-base mismatch, the photodamage time constant was higher for the perfectly complementary target sequences than for the mismatch sequences, indicating that these probes are specific for each target sequence. In addition, time constants obtained for oligonucleotide target sequences with both stem and loop base mismatches are lower than those with only loop mismatches, suggesting that these sequences are also specifically distinguished by the smart probes. These probes thus constitute robust, sensitive, specific, and cheaper alternatives to MBs for sequence-specific DNA damage detection.     

Sequence‐Dependent dsDNA‐Templated Formation of Fluorescent Copper Nanoparticles

There are only a few systematic rules about how to selectively control the formation of DNA‐templated metal nanoparticles (NPs) by varying sequence combinations of double‐stranded DNA (dsDNA), although many attempts have been made. Herein, we develop a facile method for sequence‐dependent formation of fluorescent CuNPs by using dsDNA as templates. Compared with random sequences, AT sequences are better templates for highly fluorescent CuNPs. Other specific sequences, for example, GC sequences, do not induce the formation of CuNPs. These results shed light on directed DNA metallization in a sequence‐specific manner. Significantly, both the fluorescence intensity and the fluorescence lifetime of CuNPs can be tuned by the length or the sequence of dsDNA. In order to demonstrate the promising practicality of our findings, a sensitive and label‐free fluorescence nuclease assay is proposed.     

Influence of a terminal formamido group on the sequence recognition of DNA by polyamides

Pyrrole (Py)-imidazole (Im)-containing polyamides bind in the minor groove of DNA and can recognize specific sequences through a stacked antiparallel dimer. It has been proposed that there are two different low energy ways to form the stacked dimer and that these are sensitive to the presence of a terminal formamido group: (i) a fully overlapped stacking mode in which the N-terminal heterocycles of the dimer stack on the amide groups between the two heterocycles at the C-terminal and (ii) a staggered stacking mode in which the N-terminal heterocycles are shifted by approximately one unit in the C-terminal direction (Structure 1997, 5, 1033-1046). Two different DNA sequences will be recognized by the same polyamide stacked in these two different modes. Despite the importance of polyamides as sequence specific DNA recognition agents, these stacking possibilities have not been systematically explored. As part of a program to develop agents that can recognize mismatched base pairs in DNA, a set of four polyamide trimers with and without terminal formamido groups was synthesized, and their interactions with predicted DNA recognition sequences in the two different stacking modes were evaluated. Experimental difficulties in monitoring DNA complex formation with polyamides were overcome by using surface plasmon resonance (SPR) detection of the binding to immobilized DNA hairpin duplexes. Both equilibrium and kinetic results from SPR show that a terminal formamido group has a pronounced effect on the affinity, sequence specificity, and rates of DNA-dimer complex formation. The formamido polyamides bind preferentially in the staggered stacking mode, while the unsubstituted analogues bind in the overlapped mode. Affinities for cognate DNA sequences increase by a factor of around 100 when a terminal formamido is added to a polyamide, and the preferred sequences recognized are also different. Both the association and the dissociation rates are slower for the formamido derivatives, but the effect is larger for the dissociation kinetics. The formamido group thus strongly affects the interaction of polyamides with DNA and changes the preferred DNA sequences that are recognized by a specific polyamide stacked dimer.     

CANADA: Designing nucleic acid sequences for nanobiotechnology applications

The design of nucleic acid sequences for a highly specific and efficient hybridization is a crucial step in DNA computing and DNA‐based nanotechnology applications. The CANADA package contains software tools for designing DNA sequences that meet these and other requirements, as well as for analyzing and handling sequences. CANADA is freely available, including a detailed manual and example input files, at http://ls11‐www.cs.uni‐dortmund.de/molcomp/downloads . © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Aligned carbon nanotube-DNA electrochemical sensors

Single-strand DNA chains were chemically grafted onto aligned carbon nanotube electrodes, leading to novel aligned carbon nanotube-DNA sensors of a high sensitivity and selectivity for probing complementary DNA and target DNA chains of specific sequences.     

Universal pH‐Responsive and Metal‐Ion‐Free Self‐Assembly of DNA Nanostructures

pH‐responsiveness has been widely pursued in dynamic DNA nanotechnology, owing to its potential in biosensing, controlled release, and nanomachinery. pH‐triggering systems mostly depend on specific designs of DNA sequences. However, sequence‐independent regulation could provide a more general tool to achieve pH‐responsive DNA assembly, which has yet to be developed. Herein, we propose a mechanism for dynamic DNA assembly by utilizing ethylenediamine (EN) as a reversibly chargeable (via protonation) molecule to overcome electrostatic repulsions. This strategy provides a universal pH‐responsivity for DNA assembly since the regulation originates from externally co‐existing EN rather than specific DNA sequences. Furthermore, it endows structural DNA nanotechnology with the benefits of a metal‐ion‐free environment including nuclease resistance. The concept could in principle be expanded to other organic molecules which may bring unique controls to dynamic DNA assembly.     

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.     

Target recycling amplification for sensitive and label-free impedimetric genosensing based on hairpin DNA and graphene/Au nanocomposites

The presence of exonuclease III leads to direct recycling and reuse of the target DNA, which in turn results in substantial signal amplification for highly sensitive, label-free impedimetric detection of specific DNA sequences.     

Indexing scheme and similarity measures for macromolecular sequences

Nucleotide composition and distribution along a DNA sequence is known to play a vital role in the determination of gene functions. Protein coding regions, regulatory sequences, and other functional regions are determined generally by homology studies with comparable genes from other species or specific experimental verification. With the rapid and explosive increase in sequence information, new computational techniques for rapid determination of such information and comparative studies of different genes are becoming necessary which ideally should encompass not only DNA sequences but other macromolecular sequences as well.     

Mechanism of DNA-drug interactions

Over the last two decades many strategies have been planned to design specific drugs for rare diseases to target their action at the DNA level. Advancements in our understanding of the interactions of small nonpeptide molecules with DNA have opened the doors for “rational” drug design. Special methods have now been developed to give accurate account of the precise location of ligand-DNA adducts on target DNA. We are now in a position to think of designing ligands that recognize particular sequences of base pairs. This work will allow us to enter into a new era of gene therapy for diseases like Cystic fibrosis, Alzheimer’s disease and many related disorders at genetic level. These ligands can also be employed in the treatment of various types of cancers. They may also be useful as highly specific probes to locate particular sequences in the genomic DNA.     

DNA sequence-enabled reassembly of the green fluorescent protein

We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.     

Chromosomal organization of simple repeated DNA sequences used for DNA fingerprinting

Stretches of short, simple DNA sequences are widespread in all eukaryote genomes studied so far. Simple sequences are thought to undergo frequent expansion and deletion due to intrinsic genomic mechanisms. Some of the simple sequences were used successfully to detect hypervariable loci in various genomes. Hybridization experiments using synthetic probes not only revealed the informative simple repeats suitable for DNA fingerprinting in a particular species, but also reflected the wide range of distribution of the simple sequences among eukaryotes. The organization of these simple repetitive sequences at the chromosomal loci was investigated using in situ hybridization with chemically synthesized, pure oligonucleotide probes. Both biotin- and digoxigenin-attached probes detected specific chromosomal sites that are enriched in the respective simple-repeat blocks. Depending on the organism and probe used, accumulation of simple DNA sequences at individual or multiple sites on the chromosomes of different vertebrates could be demonstrated. The simple repetitive DNA sequences are located in different chromosomal regions (e.g., heterochromatin on the sex chromosomes, nucleolus organizer regions, and R-band sites), which are constrained considerably during evolution.     

A naturally logical representation for DNA primary sequences

In this paper, we (1) introduce a logical representation (LR) for DNA primary sequences; (2) show relations between LR and some other representations including the characteristic sequences of a DNA sequence, Randic's 2-D, 4-D representations, and Z-curve (a 3-D graphical representation); and (3) outline the constructions of the S/S matrix specific for a logical sequence and its 2*2 condensed matrix.     

Sequence specific fluorescence detection of double strand DNA

Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide-fluorophore conjugates in the presence and absence of duplex DNA were examined. We observe weak TMR fluorescence in the absence of DNA. Addition of >/=1:1 match DNA affords a significant fluorescence increase over equimolar mismatch DNA for each polyamide-TMR conjugate. Polyamide-fluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site contains a non-Watson-Crick DNA base pair. A ranking of the specificity of three polyamide ring pairs-Py/Py, Im/Py, and Im/Im-was established for all 16 possible base pairs of A, T, G, and C in the minor groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T.G base pair.     

An introduction to electrochemical DNA biosensors

Electrochemical DNA biosensors exploit the affinity of single-stranded DNA for complementary strands of DNA and are used in the detection of specific sequences of DNA with a view towards developing portable analytical devices. Great progress has been made in this field but there are still numerous challenges to overcome. This review for researchers new to the field describes the components of electrochemical DNA biosensors and the important issues in their design. Methods of transducing DNA binding events are discussed along with future directions for DNA biosensors.     

Sequence‐specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid‐DNA) in capillary and microfluidic electrophoretic separations

Reversible noncovalent but sequence‐dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite‐modified oligonucleotides has enabled sequence‐specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self‐assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid‐DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence‐dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information‐processing systems.