Micelle-associated endomorphin-1 has ability to bind copper in the oxidation state either Cu(II) or Cu(I).

Reduction of Cu(II) to Cu(I) in an oxidizing extracellular environment is a potential risk factor for neurodegenerative diseases, because the re-oxidation of Cu(I) to Cu(II) can be coupled to generation of reactive oxygen species. However, little is known about how the brain is protected from the copper-induced oxidative stress. In the present study, interactions of the endogenous opioid peptide endomorphin-1 (EM1, Tyr-Pro-Trp-Phe-NH2) with ionic copper were investigated. EM1 cannot bind copper with ordinary metal coordination chemistry, since the chelate complex formation of EM1 with the metal ion is inhibited by the proline residue in the second position. In the presence of SDS micelles, however, a significant quenching of fluorescence of the tryptophan side chain of EM1 was observed on addition of copper ion, either Cu(II) or Cu(I). The spectral changes of the UV absorption of the tryptophan, which are diagnostic of cation-π interaction, were also brought about by addition of copper to EM1 only in the presence of micelles. The copper-induced spectral changes of both fluorescence and UV absorption disappeared upon the substitution of Tyr1 with alanine. The obtained results indicated that EM1 binds the copper ion through the π-electrons of aromatic side chains of Tyr1 and Trp3, which are in close contact each other in the micelle-associated form. The copper-catalyzed oxidation/reduction reaction process converting dopamine to neuromelanin, which involves potentially neurotoxic intermediates, is inhibited by EM1. Owing to the ability to bind both Cu(II) and Cu(I), EM1 may have the potential to suppress the copper-mediated oxidative stress in the brain. The present results suggest an antioxidative effect of EM1, distinct from its known analgesic effect.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.