The cleavage of phosphodiester bonds within small RNA bulges in the presence and absence of metal ion catalysts †

Kaukinen, Ulla; Bielecki, Lukasz; Mikkola, Satu; Adamiak, Ryszard W.; Lönnberg, Harri

doi:10.1039/b102519h

Cited by 43 publications

(44 citation statements)

References 47 publications

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“…On the other hand, the pK a s of the aqua complexes of Cd 2+ , Ni 2+ , Co 2+ , Pb 2+ , and Zn 2+ fall within the 7-10 range (31). This fact may contribute to the previously observed nonspecific Zn 2+ -dependent hydrolysis of RNA (32)(33)(34). Third, the geometric requirements of the binding site place a limitation on the size of the bound metal ion.…”

Section: +mentioning

confidence: 99%

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

2001

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We describe a new RNA cleavage motif, found in the hammerhead ribozyme. Cleavage occurs between nucleotides G8 and A9, yielding a free 5′-hydroxyl group and a 2′,3′-cyclic phosphate. This cleavage is dependent upon divalent metal ions and is the first evidence for a metalloribozyme known to show preference for Zn 2+ . Cleavage is also observed in the presence of Ni 2+ , Co 2+ , Mn 2+ , Cd 2+ , and Pb 2+ , while negligible cleavage was detected in the presence of the alkaline-earth metal ions Mg 2+ , Ca 2+ , Sr 2+ , and Ba 2+ . A linear relationship between the logarithm of the rate and pH was observed for the Zn 2+ -dependent cleavage, which is indicative of proton loss in the cleavage mechanism, either prior to or in the rate-determining step. We postulate that a zinc hydroxide complex, bound to the known A9/G10.1 metal ion binding site, abstracts the proton from the 2′-hydroxyl group of G8, which attacks the A9 phosphate and initiates cleavage. This hypothesis is supported by a previously reported crystal structure [Murray, J. B., Terwey, D. P., Maloney, L., Karpeisky, A., Usman, N., Beigelman, L., and Scott, W. G. (1998) Cell 92, 665-673], which shows the conformation required for RNA cleavage and proximity of the 2′-hydroxyl group to the metal ion complex.The hammerhead ribozyme ( Figure 1A) is an RNA motif that catalyzes the cleavage of an RNA substrate via a transesterification reaction, producing a 2′,3′-cyclic phosphate and a 5′-hydroxyl terminus ( Figure 1B) (1). Divalent metal ions have been implicated directly in the hammerhead ribozyme cleavage mechanism (2-5), and the most popular mechanism has involved the abstraction of the 2′-hydroxyl proton at the cleavage site by a metal ion hydroxide (6). Divalent metal ions can also play a structural role in ribozyme activity by stabilizing the catalytically active tertiary conformation through coordination to heteroatoms, such as oxygen atoms or nitrogen atoms present in the nucleoside bases. The finding that the hammerhead ribozyme is active in the absence of divalent metal ions at high concentrations of monovalent metal ions calls into question the role of divalent metal ions in the mechanism of substrate cleavage (5,7,8).Divalent metal ion binding sites in the hammerhead ribozyme have been found by X-ray crystallography (9-13) and by NMR spectroscopy (14). One of the most extensively studied metal ion binding sites is the A9/G10.1 site which involves coordination of the metal ion to N7 of G10.1 and the pro-R P phosphate oxygen of A9 (Figure 2). The pro-R P oxygen of A9 has been implicated in hammerhead ribozyme function by replacement of the nonbridging, pro-R P oxygen at the A9 phosphate with sulfur. Substrate cleavage studies in the presence of Mg 2+ resulted in reduced † This work was supported by a grant from the National Institutes of Health (GM56947).

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confidence: 99%

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

2001

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“…This system also have a sequence resemblance to a real potential therapeutic target (i.e., the breakpoint of the Leukemia related M-BCR/ABL mRNA, see below). It is known that RNA bulges are more predisposed to cleavage than fully duplexed RNA [7][8][9][10] which implies an advantage for constructs that are designed to form a bulge in the target upon binding. This also provides a pocket for potential interaction with the cleaving agent and/or recognition elements.…”

Section: Introductionmentioning

confidence: 99%

2'-O-Methyloligoribonucleotide based artificial nucleases (2’-O-MeOBAN’s) cleaving a model of the leukemia related M-BCR/ABL m-RNA

Murtola¹,

Strömberg²

2008

Arkivoc

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Several 2'-O-methyloligoribonucleotide based artificial nucleases (2'-O-MeOBANs) were developed and evaluated with respect to cleavage of a model of the Leukemia related M-BCR/ABL mRNA. All constructs cleaved the target and the system forming a triadenosine bulge in the target gave the highest rate (t 1/2 8.5 h using a 1:1 ratio of 2'-O-MeOBAN to target). The system also displays catalysis with turnover of substrate present in excess.

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“…A combination of base pairing and sequence-specific non-Watson-Crick interactions with RNA structural motifs has the potential to give higher affinity and specificity in RNA recognition than WatsonCrick pairing alone. Double-stranded RNA is considerably less prone to hydroxide or metal-catalyzed cleavage than the more flexible single-stranded RNA (also as bulges or loops; Hall et al, 1996;Portmann et al, 1996;Huesken et al, 1996;Kaukinen et al, 2001;Mikkola et al, 2001). Like singlestranded RNA, these unpaired motifs can more easily undergo the rearrangement necessary for transesterification to occur.…”

Section: Introductionmentioning

confidence: 98%

Investigation of potential RNA bulge stabilizing elements

et al. 2005

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As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2'-modified nucleoside moieties (2'-O-methyl, 2'-deoxy and 2'-deoxy-2'-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G-U wobble base pair to a G-C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (DeltaT(m) + 4.6 to + 5.4 degrees C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2'-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (DeltaT(m) + 3.6 and + 2.9 degrees C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson-Crick recognition of RNA.

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The cleavage of phosphodiester bonds within small RNA bulges in the presence and absence of metal ion catalysts †

Cited by 43 publications

References 47 publications

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

2'-O-Methyloligoribonucleotide based artificial nucleases (2’-O-MeOBAN’s) cleaving a model of the leukemia related M-BCR/ABL m-RNA

Investigation of potential RNA bulge stabilizing elements

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