FUNCTIONALLY TETHERED OLIGONUCLEOTIDES

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Under the influence of torsional stress induced by supercoiling or the binding of proteins, DNA can be coerced to adopt a variety of structures that deviate substantially from the canonical B-form duplex of Watson and Crick. Although this structural plasticity of DNA is deeply intertwined with its biological function, many facets of non-canonical DNA remain poorly understood. A primary goal of this program, broadly stated, is to elucidate how DNA structure accommodates torsional stress, to determine the energetic costs of various kinds of distortion, and to probe the effects of engineered distortions on DNA recognition by proteins. One of the principal reasons for the lack of information on non-canonical structures has been that they are generally formed only in the presence of complex macromolecular assemblies, and hence they have been difficult to embody in small, structurally characterizeable systems. The proposed studies alm to overcome this problem by use of disulfide cross-linking to engineer specific kinds of non-canonical structures in synthetic oligonucleotides. These engineered molecules will then be studied with regard to structure, energetics, and recognition by proteins. The foregoing studies provide an example of how synthetic modification of DNA can be used to gain information on its structure, function, and interaction with other macromolecules. In contrast to the numerous methods currently available for site-specific attachment of tethered functionality to DNA, relatively few methods are available for introducing the same sorts of modification into RNA. As it becomes increasingly apparent that RNA-protein interactions control a wide range of important biological processes, the need for methods to manipulate RNA structure becomes ever more acute. Another aspect of the proposed studies will address this need. Namely, chemistry will be developed to permit the site-specific attachment of tethered functionality to RNA through the convertible nucleoside approach. Several applications of these functionally tethered oligoribonucleotides will be explored: (i) synthesis of a high-capacity RNA affinity column containing the TAR element of HIV- l, to be used in purification of host RNA-binding factors that contribute to HIV transcriptional activation; and (ii) synthesis of oligoribonucleotides containing a tethered photoactive group placed within the 5'-splice site of a eukaryotic mRNA, in order to identify through photoaffinity labelling protein factors that participate in RNA splicing.