The effects of sequence context on DNA curvature.

Recent experiments have exposed significant discrepancies between experimental data and predictive models for DNA structure. These results strongly suggest that DNA structural parameters incorporated in the models are not always sufficient to account for the influence of sequence context and of specific ion effects. In an attempt to evaluate these two effects, we have investigated repetitive DNA sequences with the sequence motif GAGAG.CTCTC located in different helical phasing arrangements with respect to poly(A) tracts and GGGCCC.GGGCCC sequence motifs. Methods used are ligase-mediated cyclization and gel mobility experiments along with DNase I cutting and chemical probe studies. The results provide new evidence for curvature in poly(A) tracts. They also show that the sequence context in which bending and flexible sequence elements are found is an important aspect of sequence-dependent DNA conformation. Although dinucleotide models generally have good predictive power, this work demonstrates that in some instances sequence elements larger than the dinucleotide must be taken into account, and hence it provides a starting point for the appropriate modification and refinement of existing structural models for DNA.

A DNA sequencing strategy that requires only five bases of known terminal sequence for priming.

We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.

Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies

Antibodies reactive with native double stranded DNA are characteristic of the chronic inflammatory disease systemic lupus erythematosus. Native DNA is however, a poor immunogen and the mechanism of anti-DNA antibody production is incompletely understood. Modification of DNA can increase its immunogenicity and in inflammatory disease states reactive oxygen species produced from phagocytic cells have been shown to thus modify DNA. In this study, monoclonal antibodies produced spontaneously by two mice strains with lupus-like disease were used in a competition ELISA to monitor changes to DNA induced by reactive oxygen species. Different procedures for reactive oxygen species generation were found to cause distinct and characteristic changes to DNA involving modifications of base residues, the sugar-phosphate backbone and the gross conformational structure of double-stranded DNA. In view of this, it may be possible to use these antibodies further to probe DNA and infer the source and nature of the reactive oxygen species it has been exposed to, particularly in vivo.

Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detection of interactions between biomolecules in real time. Silanization of the grating surface was successfully realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the complementary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple usability.

Long-period fibre grating based biosensor for detection of DNA hybridisation

We implement an optical biosensor using long-period fibre grating immobilised with probe DNA. It has been used to detect hybridisation of target DNA, showing a high sensitivity and reusability function.

Long-period fibre grating based biosensor for detection of DNA hybridisation

We implement an optical biosensor using long-period fibre grating immobilised with probe DNA. It has been used to detect hybridisation of target DNA, showing a high sensitivity and reusability function.

Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detection of interactions between biomolecules in real time. Silanization of the grating surface was successfully realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the complementary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple usability. © 2007 Optical Society of America.

Repetitive DNA fragments as taxonomic markers for {\it Penaeus\/} sibling taxa (Decapoda: Dendrobranchiata: Penaeidae) from the southern terminus of the Florida peninsula, U.S.A.

Sympatric populations of P. brasiliensis and P. duorarum from Biscayne Bay, Florida, revealed species-specific satellite DNA organizational patterns with the restriction endonuclease EcoRI. The species-specific satellite DNA patterns can be explained as resulting from differential amplification/deletion events having altered monomer arrays after the divergence of these two species. Two discontinuous populations of P. duorarum (Biscayne Bay and Dry Tortugas) were found to exhibit distinct EcoRI satellite fragment patterns; BamHI repetitive fragments specific to the Dry Tortugas P. duorarum population were also detected. In addition, the evolutionary conservation of the Penaeus (Farfantepenaeus) satellites was investigated. The putative conservation of sequences related to one cloned P. duorarum satellite monomer unit suggests that the FTR satellite DNA family may not only be of use as a genome tag to distinguish between sibling and cryptic Penaeus species but may also serve as a probe to better understand decapod crustacean genome organization and evolution. ^

Enzyme-induced Aggregation of Plasmonic Nanoparticles for the Development of Label-free and Ultrasensitive Detection of Bacterial DNA

Sensitive detection of pathogens is critical to ensure the safety of food supplies and to prevent bacterial disease infection and outbreak at the first onset. While conventional techniques such as cell culture, ELISA, PCR, etc. have been used as the predominant detection workhorses, they are however limited by either time-consuming procedure, complicated sample pre-treatment, expensive analysis and operation, or inability to be implemented at point-of-care testing. Here, we present our recently developed assay exploiting enzyme-induced aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. In the experiments, AuNPs are first functionalized with specific, single-stranded RNA probes so that they exhibit high stability in solution even under high electrolytic condition thus exhibiting red color. When bacterial DNA is present in a sample, a DNA-RNA heteroduplex will be formed and subsequently prone to the RNase H cleavage on the RNA probe, allowing the DNA to liberate and hybridize with another RNA strand. This continuously happens until all of the RNA strands are cleaved, leaving the nanoparticles ‘unprotected’. The addition of NaCl will cause the ‘unprotected’ nanoparticles to aggregate, initiating a colour change from red to blue. The reaction is performed in a multi-well plate format, and the distinct colour signal can be discriminated by naked eye or simple optical spectroscopy. As a result, bacterial DNA as low as pM could be unambiguously detected, suggesting that the enzyme-induced aggregation of AuNPs assay is very easy to perform and sensitive, it will significantly benefit to development of fast and ultrasensitive methods that can be used for disease detection and diagnosis.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.