GRSDB2 and GRS_UTRdb: databases of quadruplex forming G-rich sequences in pre-mRNAs and mRNAs

G-quadruplex motifs in the RNA play significant roles in key cellular processes and human disease. While sequences capable of forming G-quadruplexes in the pre-mRNA are involved in regulation of polyadenylation and splicing events in mammalian transcripts, the G-quadruplex motifs in the UTRs may help regulate mRNA expression. GRSDB2 is a second-generation database containing information on the composition and distribution of putative Quadruplex-forming G-Rich Sequences (QGRS) mapped in ∼29 000 eukaryotic pre-mRNA sequences, many of which are alternatively processed. The data stored in the GRSDB2 is based on computational analysis of NCBI Entrez Gene entries with the help of an improved version of the QGRS Mapper program. The database allows complex queries with a wide variety of parameters, including Gene Ontology terms. The data is displayed in a variety of formats with several additional computational capabilities. We have also developed a new database, GRS_UTRdb, containing information on the composition and distribution patterns of putative QGRS in the 5′- and 3′-UTRs of eukaryotic mRNA sequences. The goal of these experiments has been to build freely accessible resources for exploring the role of G-quadruplex structure in regulation of gene expression at post-transcriptional level. The databases can be accessed at the G-Quadruplex Resource Site at: http://bioinformatics.ramapo.edu/GQRS/

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs