The chemical and enzymatic synthesis of RNA oligomers for the analysis of ribozyme structure and action

Ronald Ray Breaker, Purdue University

Abstract

Automated solid phase synthesis of RNA was conducted using synthetic units bearing a new 2$\sp\prime$ hydroxyl protecting group, o-nitrobenzyloxymethyl. A number of RNA molecules have been synthesized with overall stepwise coupling efficiencies greater than 98% using standard DNA synthesis protocols. Thermal denaturation analyses and enzymatic RNA sequence determination have confirmed the integrity of the RNA products. Different self-cleaving ribozyme systems have been synthesized using this method, including a 16 nucleotide internal fragment of the Mn$\sp{2+}$-dependent ribozyme, and have proven to be catalytically active. Also, the isosteric AMP analogue 5$\sp\prime$-deoxy-5$\sp\prime$-phosphonomethyladenosine (pcA), in which the 5$\sp\prime$ oxygen is replaced by a methylene group, was prepared chemically and converted to the ADP and ATP analogues ppcA and pppcA. Efficient polymerization of ppcA has been achieved using M. luteus polynucleotide phosphorylase; the first demonstrated enzymatic formation of the 5$\sp\prime$-deoxy-5$\sp\prime$-phosphonomethylene linkage. Polymerization products of the general form A(-cA)$\sb{\rm n}$ (where -c indicates the phosphonomethylene linkage) have been synthesized and isolated for use in the determination of the effects of this particular linkage analogue on RNA structure. Phosphonomethylene RNA polymers have proven resistant to cleavage by chemical and enzymatic RNA degradation treatments which operate via a cyclizing mechanism. Thermal UV melting curves conducted on single-stranded A(-cA)$\sb7$ molecules have demonstrated that the potential for base-stacking is retained, despite the presence of the phosphonomethylene linkage. UV mixing curve analysis of A(-cA)$\sb9$ with U$\sb{10}$ has revealed that complex formation occurs with an A to U strand ratio of 1:2, identical to that found in natural A$\sb{10}\cdot 2$U$\sb{10}$ triplexes. Thermal UV melting analyses have established that the A(-cA)$\sb9\cdot 2$U$\sb{10}$ triplex has a higher T$\sb{\rm m}$, yet experiences a reduced hypochromic effect during complex formation, than that found for the A$\sb{10}\cdot 2$U$\sb{10}$ triplex. Synthesis of RNA molecules of defined sequence containing a single internal phosphonomethylene linkage was achieved using T4 RNA ligase. Using the forcing conditions required for ligation of DNA with T4 RNA ligase, the donor pCp was joined to the acceptor A-A-cA to form the product A-A-cA-Cp. This molecule contains a single phosphonomethylene linkage bracketed by normal phosphodiesters. RNA analogues of this type would be useful as non-cleavable substrate molecules for the study of protein and RNA-based phosphodiesterases.

Degree

Ph.D.

Advisors

Gilham, Purdue University.

Subject Area

Biochemistry|Organic chemistry|Molecular biology

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