The Effect of Secondary Structure on Cleavage of the Phosphodiester Bonds of RNA

Mikkola, Satu; Kaukinen, Ulla; Lönnberg, Harri

doi:10.1385/cbb:34:1:95

Cited by 63 publications

(46 citation statements)

References 113 publications

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“…Since in-line geometry provides at most a 10 to 20-fold rate acceleration over the uncatalyzed cleavage reaction, chemical attributes beyond the local secondary structure must play a critical role in RNA catalyzed phosphoryl transfer (50,51). At present, there are two proposals for how the hairpin ribozyme achieves this rate enhancement.…”

Section: Discussionmentioning

confidence: 99%

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

et al. 2005

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The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-to 250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were solved by X-ray crystallography in a resolution range between 2.3 to 2.7 Å. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° 'in-line' geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine) and G8(uridine) folded properly, but exhibited non-ideal scissile bond geometries (τ ranging from 118° to 93°) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment, and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A−1 sugar pucker. In this regard, variants A8 and U8 appeared to represent non-productive ground-states in which their 2'-OH groups mimicked the pro-R, non-bridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A−1, lending support to the hypothesis that solvent may play an important role in the reaction. † This work was supported by NIH Grant GM63162 to J.E.W. ‡ Protein Data Bank Codes for the reported structures: 1ZFR (G8), 1ZFT (G8I), 1ZFV (G8A), 1ZFX (G8U), 2BCY (G8AP), 2BB1 (G8DAP), 2BCZ (G8I/dA−1). § Present address: Rosalind Franklin School of Science and Medicine, Dept. Biochem. and Mol. Biol., 3333 Green Bay Road Rd., N. Chicago, IL 60064, USA * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 712, Rochester, New York 14642 USA. Phone: 585 273-4516; Fax: 585 275-6007; Email: Joseph_Wedekind@URMC.Rochester.edu ∥ These authors contributed equally to this work.Supporting Information Available A stereo diagram of representative electron density maps is provided for the native and position 8 variants of this study (Figure 1). A diagram comparing the G8I/2'-OMe A−1 and G8I/2'-deoxy A−1 variants is also available (Figure 2). The method for HPLC composition analysis of AP8 and DAP8 crystals is reported, as well as elution profiles for the separated hairpin ribozyme strands (Figure 3). This information is provided free of charge at http://pubs.acs.org NIH Public Access The hairpin ribozyme is a small ribozyme whose family members catalyze a reversible, sitespecific phosphodiester bond cleavage reacti...

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Section: Discussionmentioning

confidence: 99%

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

et al. 2005

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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: 99%

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 Effect of Secondary Structure on Cleavage of the Phosphodiester Bonds of RNA

Cited by 63 publications

References 113 publications

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

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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The Effect of Secondary Structure on Cleavage of the Phosphodiester Bonds of RNA

Cited by 63 publications

References 113 publications

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

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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Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,