1CNR Istituto Circuiti Elettronici Via de Marini 6,
16419 Genova
e-mail:arrigo@ice.ge.cnr.it
2E.O. Ospedali Galliera,
Via Mura delle Capuccine
3Universita' degli Studi di Genova,Dipartimento Scienze della Salute.
One of the major impact of genome sequencing project is the approach to drug discovery; there is a strong pressure to efficiently and quickly mine the huge amount of information available in the databases. The main goal of this integrated research activity is the functional characterisation of crude non-annotated sequences; it is possible to apply different experimental method in order to ascertain the biological function of a given molecular target; the subsequent step after the target identification is its validation for appropriate drug discovery. Several technologies re available for this purpose,the Antisense oligonucleotides (ASO) are one of the useful method for the experimental evaluation of the gene expression and for its inhibition by translation interference. The application of ASO techniques has acquired high relevance in the post-genomic phase; the functional characterisation of the rough genomic sequence needs quick and reliable experimental method ; this task could be easily performed by antisense oligonucleotides. The Antisense design strategy require the formation of a base pairing between the target construct and the mRNA whose function is to be suppressed; the most important factor is the Watson-Crick base pairing rule for an high selective targeting of an mRNA. The hybridisation between mRNA and ASO originated a duplex formation that prevent the ribosome scanning phase. The binding affinity of the antisense molecule is correlated to the melting temperature and the number of the hydrogen bond between the ASO and the mRNA target sequence. The optimal length of an antisense oligo is comprised between 15-20 base pair; this range of length ensure to pick up an unique sequence and also to allow the maximisation of the discriminating capability between two gene products that differ by a single base. The characterisation of the best sequences for ASO application is based on empirical approaches, the widely applied procedure requires the synthesis of oligonucleotide that target domains scattered along the entire molecule of the mRNA (Gene-walk approach). The identification of optimal ASO target domains on cDNA is a not simple task, there are not unique method for oligos design, the canonical techniques for PCR probe can fail to detect the level of hybridisation affinity; the melting temperature (Tm) is not the only parameter necessary to evaluate the hybridisation affinity of a nucleotide domain. In the perspective to optimise the design of these molecule a bioinformatic tool can help this kind of research. In this paper we have developed and applied a new software for the ASO selection based on the search of potential target by a combined approach of a clustering method with mechanical statistic ; AGeWa(Automatic Genome Walk) is able to select the candidate target on the primary sequence taking in account different biochemical properties.