Influence of sequence variation on the RNA cleavage activity of Zn²⁺-dimethyl-dppz-PNA-based artificial enzymes

Abstract

The ability of Zn2+ dimethyl-dppz PNAzymes to cleave RNA target sequences with under 20 minute half-lives is critically dependent on the bulge-closing base pairs as well as their stacking interactions with the neighbouring nucleobases.

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“…(? :A) and G:T (T:G) [37-39] may occur between olinscides and imperfect sites of rRNAs of non-target organisms. [1].…”

Zinc N,N-bis(2-picolyl)amine Chelates Show Substitution-Dependent Cleavage of Phosphodiesters in Models as Well as of PNAzyme-RNA Bulges

“…The three ligands were conjugated to the PNA sequence via standard HATU peptide chemistry (Scheme 4) and the PNA was The carboxylic acid derivatives 9, 10 and 11 were conjugated to PNA in order to synthesise the PNAzymes. The PNA oligomer was synthesized using a microwave-assisted peptide synthesizer Biotage ® Initiator+ Alstra ™ (Uppsala, Sweden) as described in the literature [8][9][10][11]. Selective deprotection of the central 4-methyltrityl protected amine with 0.1% TFA in DCM, as previously reported [8,9], resulted in the free primary amine for conjugation to the N,N-bis(2-picolyl)amine ligands.…”

“…Purification via HPLC resulted in the final PNAzymes (PNAzyme 1, H; PNAzyme 2, Me and PNAzyme 3, NH 2 ) which were characterized via mass analysis with MALDI-TOF MS and used for the kinetic studies. Concentrations of the purified PNAzymes were determined spectrophotometrically with nearest-neighbour approximation [8][9][10][11].…”

“…Most of the reactive species to date with high site selectivity use Cu(II) as the metal ion [8,9], but this presents toxicity challenges in biological systems. Zn(II) has recently been used, and with similar or even slightly higher activity [10,11]. However, further search for alternative chelates is desirable, especially if therapeutic applications are going to be realized; and this development will be assisted by an accompanying understanding of the mechanistic principles that govern activity.…”

Organometallic modification confers oligonucleotides new functionalities

“…As a rare and remarkable example, artificial DNAzyme and PNAzyme ribonucleases relying on coordination of Zn(II) have reached potentially useful levels of catalytic activity at Zn(II) concentrations close to those prevailing inside malaria-infected red blood cells. [129][130][131] However, it should be pointed out that Zn(II) is the most abundant transition metal ion in cells and utilizing any other metal ion in the same way would be much more difficult. Finally, some applications place strict demands not only on the number of available coordination sites but also on the geometry of the complex.…”