Effects of metal ions, including Mg2+ and lanthanides, on the cleavage of ribonucleotides and RNA model compounds.

Breslow, Ronald; Huang, Deeng-Lih

doi:10.1073/pnas.88.10.4080

Cited by 156 publications

(83 citation statements)

References 18 publications

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“…The degree of polymerization did not have a significant effect on the catalytic activity (entry 6). Nevertheless, polyvinylimidazoles were found to be inferior in the cleavage compared with polyethylenimine derived polymers (entries [8][9][10][11][12][13][14], which illustrated that the binding of cationic polyethylenimine backbones in polymers 3 with the anionic phosphates in poly(U)-and the likely catalytic role of the protonated backbone amino groups in proton transfer-accelerated the cleavage. That was further confirmed by a control experiment using unsubstituted polyethylenimine 2 as the catalyst (entry 7), from which a small hydrolytic acceleration was observed, indicating that the partially protonated backbone not only binds with the negative charged phosphodiester bonds through proton transfer, but also contributes slightly to the cleavage (30).…”

Section: Resultsmentioning

confidence: 99%

“…This is then hydrolyzed in subsequent steps to the final product with a phosphate group on C-3′ and a hydroxyl group back on C-2′. We have described biomimetic systems that mimic this process, using either concentrated imidazole buffer with RNA itself (6-10) or cyclodextrins carrying attached imidazole rings to cleave RNA analogs (11)(12)(13). We now describe the cleavages of poly(U) (>300 units) by two classes of synthetic polyimidazoles as ribonuclease A mimics, again forming a C-5′ hydroxyl group on the leaving RNA unit as with ribonuclease A.…”

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confidence: 99%

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Binding and biomimetic cleavage of the RNA poly(U) by synthetic polyimidazoles

Cheng

Abhilash

Breslow

2012

Proc. Natl. Acad. Sci. U.S.A.

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Four polyimidazoles were used in the binding and cleavage studies with poly(U). The two polydisperse polyvinylimidazoles were previously described by others, while the other two new polymers of polyethyleneimines were prepared by cationic polymerization of oxazolines. The latter had imidazole units attached to each nitrogen of the polymers. They were characterized by gel permeation chromatography and had very low polydispersities. When they were partially protonated they bound to the poly(U) and catalyzed its cleavage by a process analogous to that used by the enzyme ribonuclease A. The kinetics of the cleavage were followed by an assay we had previously described using phosphodiesterase I from Crotalus venom after the cleavage processes. Cleavage of poly(U) with Zn 2+ was also examined, with and without the polymers. A scheme is described in which these cleavages could be made sequence selective with various RNAs, particularly with important targets, such as viral RNAs.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Binding and biomimetic cleavage of the RNA poly(U) by synthetic polyimidazoles

Cheng

Abhilash

Breslow

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…In this study, sterile RNAse and DNAse-free water was chosen as the medium of RNA elution, as the pH is around 7-7.5, maintaining the reactivity of the 2 0 -OH at very low level, and to ensure the absence of other metal ions such as Mg 2? that can mediate the cleavage of RNA, promoting RNA degradation (Breslow and Huang 1991). In this experiment, since the RNA product has the size of 77nt, this RNA product was used as the experimental subject to mimic the elution of small RNAs from denaturing polyacrylamide gel which are within the range of 21-26 nucleotides long.…”

Section: Resultsmentioning

confidence: 99%

A rapid and cost effective method in purifying small RNA

Citartan

Tan

Tang

2011

World J Microbiol Biotechnol

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Purification of RNA fragments from a complex mixture is a very common technique, and requires consideration of the time, cost, purity and yield of the purified RNA fragments. This study describes the fastest method of purifying small RNA with the lowest cost possible, without compromizing the yield and purity. The technique describes the purification of small RNA from polyacrylamide gel, resulting in a good yield of small RNA with minimum experimental steps in avoiding degradation of the RNA, obviating the use of ethidium bromide and phenol-chloroform extraction, as well as siliconized glass wools to remove the polyacrylamide gel particles. The purified small RNA is suitable for a wide variety of applications such as ligation, end labelling with radio isotope, RT-PCR (Reverse Transcriptase-PCR), Northern blotting, experimental RNomics study and also Systematic Evolution of Ligands by Exponential Enrichment (SELEX).

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“…3), showing that the cleavage by 1 involved functional imidazole groups along with the polycation backbone (24,25). On the other hand, with the natural (not deoxygenated) polypeptide poly-L-histidine·HCl, which shares the same chiral configuration (L-) with DOPP 1 but lacks positive charged secondary amino groups on the backbone, almost no acceleration was observed ( (26,27) and by rare earth ions, such as Eu 3+ and Yb 3+ (16). Thus, we have also reexamined the system and extended it to a number of metal ions in 1:1 combination with DOPP 1 for the cleavage of poly(U) ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…A likely sequence for this second step is protonation of the O − on the right by the BH + with deprotonation of the OH group on the left by B: to form a new monoanion and a new BH + , then proton transfer of that BH + to the leaving oxygen which is expelled by the oxyanion on the left forming a P=O double bond, and then proton reabstraction on the right to form the cyclic anion. Note that the cyclic anion must be formed with B: on the left and BH + on the right to explain why the cyclic phosphate is more slowly hydrolyzed as an added substrate by RNase A, with the reverse protonation state, than it is when it is formed by this process (8,16). The hydrolysis must proceed by a reverse of the cyclization, with H 2 O replacing the departed leaving group.…”

Section: Significancementioning

confidence: 99%

Deoxypolypeptides bind and cleave RNA

Cheng

Mahendran

Gonzalez

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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We have prepared L-and D-deoxypolypeptides (DOPPs) by selective reduction of appropriately protected polyhistidines with borane, reducing the carbonyl groups to methylenes. The result is a chiral polyamine, not amide, with a mainly protonated backbone and chirally mounted imidazolylmethylene side chains that are mostly unprotonated at neutrality because of the nearby polycationic backbone. We found that, in contrast with the D-octahistidine DOPP, the L-octahistidine DOPP is able to cooperatively bind to a D-polyuridylic acid RNA; this is consistent with results of previous studies showing that, relative to D-histidine, L-histidine is able to more strongly bind to RNA. The L-DOPP was also a better catalyst for cleaving the RNA than the D-DOPP, consistent with evidence that the L-DOPP uses its imidazole groups for catalysis, in addition to the backbone cations, but the D-DOPP does not use the imidazoles. The L-DOPP bifunctional process probably forms a phosphorane intermediate. This is a mechanism we have proposed for models of ribonuclease cleavage and for the ribonuclease A enzyme itself, based on our studies of the cleavage and isomerization of UpU catalyzed by imidazole buffers as well as other relevant studies. This mechanism contrasts with earlier, generally accepted ribonuclease cleavage mechanisms where the proton donor coordinates with the oxygen of the leaving group as the 2-hydroxyl of ribose attacks the unprotonated phosphate. T he enzyme ribonuclease A (RNase A) hydrolyzes RNA with bifunctional catalysis, using His-12 as a base and protonated His-119 as an acid to form the first products, a 2,3-cyclic phosphate of one ribose and the free next ribose with a new terminal 5′-hydroxyl group (1, 2). Lys-41 is also involved in stabilizing intermediate anions (3). In a second step the cyclic phosphate is hydrolyzed using the now-protonated His-12 and now-deprotonated His-119 in essentially a reverse of the cyclization step, with water replacing the second ribose unit (4). In the classical mechanism of this process it had been generally accepted, including in textbooks, that the protonated His-119 donates its proton to the leaving group whereas the 2′-hydroxyl group attacks the phosphate (1, 2) [isotope studies show that two protons are "in flight" during the hydrolysis step, essentially the reverse of the cyclization, both moving at the same time (5)]. We also showed the two-proton-in-flight process in a model system (6).However, our extensive studies on the cleavage of RNA with imidazole-imidazolium catalysts and on catalysis by RNase A itself indicated that a different mechanism is used ( Fig. 1) (7, 8). The proton donor BH + hydrogen binds to a phosphate oxyanion and donates the proton to the phosphate as the ribose 2′-hydroxyl adds while losing a proton to base :B. This process results in an intermediate phosphorane species. This then expels the 5′-hydroxyl group of the second nucleotide to cleave the P-O bond in a second step. We proposed this two-step phosphorane process to be the preferred path for th...

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Effects of metal ions, including Mg2+ and lanthanides, on the cleavage of ribonucleotides and RNA model compounds.

Cited by 156 publications

References 18 publications

Binding and biomimetic cleavage of the RNA poly(U) by synthetic polyimidazoles

Binding and biomimetic cleavage of the RNA poly(U) by synthetic polyimidazoles

A rapid and cost effective method in purifying small RNA

Deoxypolypeptides bind and cleave RNA

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