Deoxypolypeptides bind and cleave RNA

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.The 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 the enzyme, a proposal further supported by computation (9–11). For a general review of phosphoranes as well-demonstrated intermediates in phosphate reactions, see ref. 12. We believe the phosphorane mechanism likely for the enzyme RNase A is essentially the same as that for some artificial enzymes, in which a simultaneous bifunctional mechanism operates in an enzyme mimic. This was demonstrated in one of our model studies (8).Open in a separate windowFig. 1.Combination of a base B: and an acid BH⁺ converts a uridine monomer to its phosphorane by a simple shift of two protons. In the second step the formed B: helps transfer a proton to the leaving group oxygen from the new OH group to form the cyclic phosphate and the new R-CH₂-OH.There are two reasons to create enzyme mimics: (i) to create new useful catalysts, and (ii) to gain insight into the enzymatic process itself. Ideally one would want to use polyhistidine as an artificial enzyme to cleave RNA, and use sequence recognition to preferentially cleave RNAs that cause disease, but polyhistidine is too insoluble in water at physiological pH to serve this role (13). We previously described a simple polyethylenimine with imidazole groups linked to the nitrogens through methylene chains that indeed catalyzed the cleavage of a polyuridylic acid [poly(U)] RNA (14). The polymer backbone nitrogens were mainly protonated at neutrality whereas the imidazoles were mainly unprotonated because of the cationic charge of the backbone. As with RNase A, we used the imidazole ring of the polymer as the base, but chose the more basic backbone amino groups as the protonated acid catalysts. The enzyme uses a protonated imidazole for this step, from a histidine made more basic by its protein environment, a complexity that is not feasible in a model system. This work was aimed at creating a catalyst that, with sequence recognition, could selectively cleave a viral RNA in a medically useful way, goal 1.Now we have designed and prepared new chemically defined polyamines from synthetic chiral polypeptides, histidine octamers, using our previously described process in which polypeptides can be deoxygenated with borane (15). This reaction preserves the chirality of the histidine units, so we compared the octamer derived from L-histidine with that from D-histidine to get further insight into the details of the catalytic process, goal 2. We now see that the L-deoxypolypeptide (L-DOPP) 1 binds cooperatively to poly(U), and is also a better catalyst for poly(U) cleavage, than is the D-enantiomer (D-DOPP 2). The l-DOPP uses a bifunctional mechanism in which a simple shift of two protons in two hydrogen bonds would lead to the phosphorane (Fig. 1), whereas the DOPP 2 uses principally the backbone nitrogens, probably with one as B: and another as BH⁺.This work supports our mechanism for RNase A that proceeds through a phosphorane intermediate, not the classical mechanism that involves direct protonation of the leaving group while the nucleophilic 2-OH group is attacking. Protonation of the leaving group is a transfer catalyzed by the backbone amine in our process, or in the enzyme by His-119, and is a second step performed on the phosphorane intermediate. 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 H₂O replacing the departed leaving group.     

Paired exchange programmes can expand the live kidney donor pool

BACKGROUND: Kidney paired donation (KPD) is an exchange of organs between two live donors, who are otherwise ABO incompatible or cross-match positive, and their intended recipients. The outcome is the generation of compatible transplants conferring an improvement in quality of life and longevity. METHODS: Medline was searched for articles on KPD using a combination of keywords. Publications focusing on protocols and policy, mathematical modelling, ethical controversies, and legal and logistical barriers were identified. RESULTS: Many are precluded from transplantation because of incompatibilities with their intended donors. KPD has the potential to increase the rate of transplantation by facilitating exchange transplants between otherwise incompatible donor-recipient couples. Ethical controversies surrounding paired donation include confidentiality, conditionality of donation, synchronicity of operations and the possibility of disadvantaging blood group O recipients. Logistical barriers hampering KPD programmes involve the location of donor surgery and organ transport. CONCLUSION: Paired donation may expand the living donor pool by providing an alternative successful strategy for incompatible donor-recipient couples. Its widespread implementation will depend on resolving ethical and logistical constraints.     

Ongoing quality monitoring and enhancement explained

The University of Northampton (formerly University College Northampton) was one of five sites selected to pilot ongoing quality monitoring and enhancement (OQME). The process measures the quality of healthcare education programmes provided by academic institutions and clinical learning environments using an integrated and streamlined system. This article describes OQME from a clinical placement perspective.