A method of analysis is presented that utilizes matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and products of RNA cleavage, by use of a program designed to mass-match observed MS peaks with predicted RNA cleavage products. The method is illustrated through application to the study of targeted oxidation of RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by metallonucleases. Following incubation of each RNA with catalysts and/or redox co-reactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full-length RNA. For each RNA, a unique list was generated that contained the predicted masses of both the full-length, and all of the possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of the six expected overhangs formed at nascent termini adjacent to the cleavage sites. The overhangs corresponded to 2′,3′-cyclic phosphate, 3′-phosphate, 3′-phosphoglycolate, 5′- hydroxyl and 5′- phosphate, which corresponded to differing oxidative, hydrolytic, and/or 2′-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a mass-matching tolerance of 200 ppm. Both time-dependent cleavage mediated by metallonucleases and MALDI-TOF-induced fragmentation were observed, and these were distinguished by time-dependent experiments. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang at each nucleotide position. Limitations included artifactual skewing of quantification by mass bias, a limited mass range for quantification, and a lack of detection of secondary cleavage products. Nevertheless, the method presented herein provides a rapid, accurate, highly-detailed and semi-quantitative analysis of RNA cleavage that should be widely applicable.

Catalytic metallodrugs were used to oxidatively cleave HIV-1 Rev Response Element RNA (RRE RNA), and the mechanisms of RNA cleavage were studied using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), fluorescence spectroscopy, and gel electrophoresis. The metallodrugs, which contained combinations of the transition metals Fe2+, Co2+, Ni2+, and Cu2+ and the Rev-coupled chelators DOTA, DTPA, EDTA, NTA, tripeptide GGH, and tetrapeptide KGHK, bind to and cleave HIV RRE RNA through heretofore unknown oxidative mechanisms. The broad spectrum of metal catalysts and co-reagents provided a means for systematic variation of oxidative reactivity without significant perturbation of binding between catalyst and RNA. Detailed MS analyses were used to monitor formation of RNA fragments containing terminal 2’,3’-cyclic phosphate (2’,3’-cPO4), 3’-phosphate (3’-PO4), 3’-phosphoglycolate (3’-PG), 5’- hydroxyl (5’-OH), 5’- phosphate (5’-PO4) and other nascent overhangs at sites of cleavage. The distinct overhangs corresponded to distinct mechanisms of oxidative hydrogen-abstraction (H abstraction), hydrolysis, and/or endonucleolysis, allowing a dissection of the contributions of various mechanisms of oxidative cleavage. Rapid co-reactant- and catalyst-dependent formation of fragments containing terminal 3’-PG, 3’-PO4 and 5’-PO4 overhangs appeared to be initiated primarily by H abstraction events. The standard thiobarbituric acid (TBA) assay was employed herein in a novel usage to monitor the formation of base 2-hydroxypropenal products produced by 4’-H abstraction in RNA. Formation of an adduct with TBA was monitored by fluorescence, and its quantification correlated with the formation of 3’-PG monitored by MALDI-TOF MS, confirming oxidative 4’-H abstraction as a major mechanism of rapid catalyst-mediated cleavage of RRE RNA. Rapid formation of 3’-PO4 overhangs was most likely a result of 5’-H abstraction. Apparent rates of formation of 3’-PG (a unique product of 4’-H abstraction) at differing nucleotide positions within the RNA were used to triangulate probable 3D positions of metal centers and establish the distance-dependence of 4’-H abstraction for certain catalytic metallodrugs.

Artificial nucleases containing Rev-coupled metal chelates based on combinations of the transition metals Fe(2+), Co(2+), Ni(2+), and Cu(2+) and the chelators DOTA, DTPA, EDTA, NTA, tripeptide GGH, and tetrapeptide KGHK have been tested for DNA nuclease activity. Originally designed to target reactive transition metal chelates (M-chelates) to the HIV-1 Rev response element mRNA, attachment to the arginine-rich Rev peptide also increases DNA-binding affinity for the attached M-chelates. Apparent K(D) values ranging from 1.7 to 3.6 µM base pairs for binding of supercoiled pUC19 plasmid DNA by Ni-chelate-Rev complexes were observed, as a result of electrostatic attraction between the positively-charged Rev peptide and negatively-charged DNA. Attachment of M-chelates to the Rev peptide resulted in enhancements of DNA nuclease activity ranging from 1-fold (no enhancement) to at least 13-fold (for Cu-DTPA-Rev), for the rate of DNA nicking, with second order rate constants for conversion of DNA(supercoiled) to DNA(nicked) up to 6 × 10(6) M(-1) min(-1), and for conversion of DNA(nicked) to DNA(linear) up to 1 × 10(5) M(-1) min(-1). Freifelder-Trumbo analysis and the ratios of linearization and nicking rate constants (k(lin)/k(nick)) revealed concerted mechanisms for nicking and subsequent linearization of plasmid DNA for all of the Rev-coupled M-chelates, consistent with higher DNA residency times for the Rev-coupled M-chelates. Observed rates for Rev-coupled M-chelates were less skewed by differing DNA-binding affinities than for M-chelates lacking Rev, as a result of the narrow range of DNA-binding affinities observed, and therefore relationships between DNA nuclease activity and other catalyst properties, such as coordination unsaturation, the ability to consume ascorbic acid and generate diffusible radicals, and the identity of the metal center, are now clearly illustrated in light of the similar DNA-binding affinities of all M-chelate-Rev complexes. This work paints a clearer picture of the factors governing DNA nuclease activity by redox active M-chelates than was previously possible. The results demonstrate enhancement of DNA cleavage by use of a targeting sequence, but also clearly underscore that significant orientational factors are required for optimal reactivity at the metal center. Moreover, the studies confirm high selectivity for the target HIV RRE RNA at the most likely dosage concentrations, lending further support to the feasibility of designing and applying targeted catalytic metallodrugs.