DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications. The sensitivity and selectivity of multiplexed single-stranded DNA (ssDNA) surface bioaffinity sensing can be greatly enhanced when coupled to a surface enzymatic reaction. Herein we describe a novel method where the specific sequence-dependent adsorption of a target ssDNA template molecule onto a ssDNA-modified gold microarray is followed with the generation of multiple copies of ssRNA via in situ surface transcription by RNA polymerase. The RNA created on this “generator” element is then detected by specific adsorption onto a second adjacent “detector” element of ssDNA that is complementary to one end of the ssRNA transcript. SPR imaging is then used to detect the subsequent hybridization of complementary DNA-coated gold nanoparticles with the surface-bound RNA. This RNA transcription-based, dual element amplification method is used to detect ssDNA down to a concentration of 1 fM in a volume of 25 μL (25 zeptomoles).

The Protein Structure Initiative’s Structural Biology Knowledgebase (SBKB, URL: http://sbkb.org) is an open web resource designed to turn the products of the structural genomics and structural biology efforts into knowledge that can be used by the biological community to understand living systems and disease. Here we will present examples on how to use the SBKB to enable biological research. For example, a protein sequence or Protein Data Bank (PDB) structure ID search will provide a list of related protein structures in the PDB, associated biological descriptions (annotations), homology models, structural genomics protein target status, experimental protocols, and the ability to order available DNA clones from the PSI:Biology-Materials Repository. A text search will find publication and technology reports resulting from the PSI’s high-throughput research efforts. Web tools that aid in research, including a system that accepts protein structure requests from the community, will also be described. Created in collaboration with the Nature Publishing Group, the Structural Biology Knowledgebase monthly update also provides a research library, editorials about new research advances, news, and an events calendar to present a broader view of structural genomics and structural biology.

DNA microarrays are invaluable tools for biosensing applications such as diagnostic detection of DNA and analysis of gene expression. Surface plasmon resonance imaging (SPRI) can detect unlabeled oligonucleotide targets adsorbed to the array elements. The variety of biosensing applications can be expanded by enzymatic manipulation of DNA microarray elements and the sensitivity of detection can be enhanced with the use of oligonucleotide immobilized onto a gold nanoparticle surface (detector-NP). We describe a novel method that couples a template-directed polymerase extension of a surface array element with nanoparticle-enhanced detection of the reaction product. Using this technique, it is possible to see as little as 10–100 attomoles of polymerase product representing as little as 0.25% of a monolayer. This sensitivity would allow for the detection of a specific DNA target that is present in low amounts in a sample and with partially unknown sequence. One application of this method would be to identify the presence of the aberrantly recombined DNA sequences, such as those found in the fragile sites of chromosomes.

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We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.