Synthesis of High Density RNA Miccroarrays

The purpose of this project is to develop a powerful new method for synthesizing RNA and DNA/RNA chimeric microarrays for applications in fundamental biochemical research, and for possible future applications in aptamer research for nanotechnology-based diagnosis and therapy. The RNA microarrays will be synthesized in situ on glass substrates using the recently developed RNA phosphoramidite monomers incorporating acetal levulinyl ester (ALE) 2`-hydroxyl protecting groups, and levulinyl (Lv) and dimethylformamidine (DMF) base protecting groups. Microarray layout will be directed by selective removal of the 5`-nitrophenylpropyloxycarbonyl (NPPOC) photocleavable protecting group on the RNA phosphoramidites by a digital micromirror ultraviolet photolithographic system. For this project, up to 780 thousand unique RNA sequences for the detection of RNA binding proteins will be included in single RNA microarrays or RNA/DNA chimeric microarrays. The primary application of RNA or DNA/RNA chimeric microarrays will initially be in molecular cell biology, for the study of post-transcriptional regulation of gene expression. Post-transcriptional regulation, by means of RNA binding proteins is now known to play a key role in gene expression, however the mechanisms are varied, complex and incompletely understood. The initial RNA microarrays will be used to understand the sequence dependent binding affinity for the FBF-2 PUF protein from Caenorhabditis elegans. This protein binds the 3` untranslated region of target messenger RNAs to regulate translation and mRNA stability. This mechanism controls the hermaphroditic germline switch from spermatogenesis to oogenesis, as well as the maintenance of germline stem cells. The PUF protein-binding RNA microarray will determine binding affinity sequence specificity by including all 49 (262,144) permutations of the nine base core protein-binding region of the wild-type gld-1 FBEa 28mer mRNA sequence. The project also encompasses the synthesis of RNA/DNA chimeric microarrays using NPPOC protected RNA and DNA phosphoramidites. These RNA/DNA chimeric microarrays are intended to probe the 2`-hydroxyl recognition mechanism of the FBF-2 PUF protein by including all possible single and multiple sequence-preserving RNA to DNA mutations of the core binding element of gld-1 FBEa.

The purpose of this project was to develop a powerful new method for synthesizing RNA and DNA/RNA chimeric microarrays for applications in fundamental biochemical research, and for possible future applications in aptamer research for nanotechnology-based diagnosis and therapy.The RNA microarrays are synthesized in situ on glass substrates using the developed RNA phosphoramidite monomers incorporating acetal levulinyl ester (ALE) 2?-hydroxyl protecting groups, and levulinyl (N-Lev) and dimethylformamidine (N-DMF) base protecting groups. Microarray layout is directed by selective removal of the 5?-nitrophenylpropyloxycarbonyl (NPPOC) photocleavable protecting group on the RNA phosphoramidites by a digital micromirror ultraviolet photolithographic system. As a result of this project, we can now synthesize, on single glass surface with an area of 1.4 cm2, up to 780 thousand unique RNA sequences. We use these RNA sequences for the detection of RNA binding proteins and other affinity and enzymatic interactions specific to RNA. In addition, we can combine DNA and RNA synthesis chemistry in order to synthesize RNA/DNA chimeric microarrays that we use to understand how RNA binding proteins and enzymes distinguish between RNA and DNA.The primary application of RNA or DNA/RNA chimeric microarrays will initially be in molecular cell biology, for the study of post-transcriptional regulation of gene expression. Post-transcriptional regulation, by means of RNA binding proteins is now known to play a key role in gene expression, however the mechanisms are varied, complex and incompletely understood. We are using the initial RNA microarrays to understand the sequence dependent binding affinity for the FBF-2 PUF protein from Caenorhabditis elegans. This protein binds the 3 untranslated region of target messenger RNAs to regulate translation and mRNA stability. This mechanism controls the hermaphroditic germline switch from spermatogenesis to oogenesis, as well as the maintenance of germline stem cells. The PUF protein-binding RNA microarray will determine binding affinity sequence specificity by including all 49 (262,144) permutations of the nine base core protein-binding region of the wild-type gld-1 FBEa 28mer mRNA sequence. In addition we make RNA/DNA chimeric microarrays by using both NPPOC protected RNA and DNA phosphoramidites. We use these RNA/DNA chimeric microarrays to probe the 2?-hydroxyl recognition mechanism of the FBF-2 PUF protein by including all possible single and multiple sequence-preserving RNA to DNA mutations of the core binding element of gld-1 FBEa.