To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.

Knowledge about the structure of RNA is crucial to understanding its biological activities. Very often, the presence of unusually thermodynamically stable structural fragments in RNAs, such as hairpins, makes it impossible to apply primer extension to visualize the results of chemical mapping experiments. However, replacement of DNA primers with LNA-modified primers overcomes this limitation. This approach was tested successfully on regulatory OxyS RNA and DsrA RNA from Escherichia coli.

Information on the secondary structure and interactions of RNA is important to understand the biological function of RNA as well as in applying RNA as a tool for therapeutic purposes. Recently, the isoenergetic microarray mapping method was developed to improve the prediction of RNA secondary structure. Herein, for the first time, isoenergetic microarrays were used to study the binding of RNA to protein or other RNAs, as well as the interactions of two different RNAs and protein in a three-component complex. The RNAs used as models were the regulatory DsrA and OxyS RNAs from Escherichia coli, the fragments of their target mRNAs (fhlA and rpoS), and their complexes with Hfq protein. The collected results showed the advantages and some limitations of microarray mapping.