There are a growing number of RNAs for which biological function and structure are not known. The knowledge of RNA structure is crucial to the understanding of its biological function. It is also helpful for applying RNA as a potential agent or target for therapeutic treatment. The structure dynamics of RNA is important for biological function and enables to interfere with the activity of RNA. Novel isoenergetic microarrays have been applied to study the structure and interactions of RNA.

In native RNA, single stranded regions are short (4-6 nucleotides on average) and are often involved in weak non-Watson-Crick and tertiary interactions. Most regions of RNA interact with complementary strands and are unable to bind to the single stranded probes on RNA microarrays. RNA microarrays built with short (5-6 mers) of highly modified RNA probes that are covalently linked to agarose coated microscope slides are used. The 2’-O-methylated and LNA-modified oligomer probes have been designed in such a way, that the interaction of each probe with its complementary single stranded fragment of target RNA has the same thermodynamic stability (is isoenergetic). The thermodynamic stabilities of modified probes binding to complementary fragments of target RNA were predicted based on the measurement of the thermodynamic stabilities of many model LNA-2’OMeRNA/RNA duplexes. A library of 853 isoenergetic pentamers (from 1024 possible pentamers) was chemically synthesized, deprotected and purified.

The RNA microarray method was tested and optimized on several natural RNA. The model target RNA was the 5S rRNA from E. coli (nonisoenergetic probes). Experiments were also performed on the 323 nucleotide R2 5’RNA from Bombyx mori (with isoenergetic probes), whose structure was unknown. The hybridization results were used as constrains in the RNAstructure program to predict RNA secondary structure. In the case of 5S rRNA, microarray experiments allowed for 93% correct prediction of base pairing. Microarray experiments on the Bombyx mori R2 5’RNA lead to the proposal of a secondary structure which is consistent with the one determined from chemical mapping experiments. Isoenergetic RNA microarrays have been also applied for prediction of structures of R2 5’RNAs from Bombyx pyri, Bombyx prometheus, Bombyx hercules, and Bombyx cynthia as well as OxyS RNA and DsrA RNA from E. coli. RNA microarrays have been used for some of these target RNAs also for study RNA-protein interactions.

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