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Isolation of a Ribozyme with 5'-5' Ligase Activity

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Isolation of a Ribozyme with 5'-5' Ligase Activity View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Isolation of a ribozyme with 5’-5’ ligase activity Karen B Chapman+ and Jack W Szostak* Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA Background: Many new ribozymes, including sequ- linkage. Deletion analysis of one of the selected ence-specific nucleases, ligases and kinases, have been sequences revealed that a 54-nucleotide RNA retained isolated by in vitro selection from large pools of random- activity; this small ribozyme folds into a pseudoknot sec- sequence RNAs.We are attempting to use in vitro selec- ondary structure with an internal binding site for the tion to isolate new ribozymes that have, or can be substrate oligonucleotide.The ribozyme can also synthe- evolved to have, RNA polymerase-like activities. As size 5’-5’ triphosphate and 5’-5’ pyrophosphate linkages. phosphorimidazolide-activated nucleosides are exten- Conclusions: The emergence of ribozymes that acceler- sively used to study non-enzymatic RNA replication, we ate an unexpected 5’-5’ ligation reaction from a selection wished to select for a ribozyme that would accelerate the designed to yield template-dependent 3’-5’ ligases template-directed ligation of 5’-phosphorimidazolide- suggests that it may be much easier for RNA to catalyze activated oligonucleotides. the synthesis of 5’-5’ linkages than 3’-5’ linkages. 5’-5’ Results: Ribozymes selected to perform the desired linkages are found in a variety of contexts in present-day template-directed ligation reaction instead ligated them- biology. The ribozyme-catalyzed synthesis of such selves to the activated substrate oligonucleotide via their linkages raises the possibility that these 5’-5’ linkages 5’-triphosphate, generating a 5’-5’ P’,P4-tetraphosphate originated in the biochemistry of the RNA world. Chemistry & Biology May 1995, 2:325-333 Key words: evolution, in vitro selection, origin of life, RNA world introduction these ribozymes conserves the total number of phospho- We have been pursuing the discovery of new ribozymes diester bonds. The joining reaction is therefore largely to provide an experimental foundation for thinking driven entropically, by the release of guanosine, and the about the potential functions of ribozymes in early cells, accumulation of this product could ultimately limit the and as a way of providing new starting points for replication of longer templates. Recently, we have iso- attempts to evolve self-replicating RNAs. In vitro selec- lated ribozymes that catalyze the ligation of oligonu- tion has proven to be a powerful method for the isola- cleotides with the same chemistry used by present-day tion of RNAs with novel functions from large pools of polymerases, that is, the attack of a 3’ hydroxyl on the random sequences [l]. In vitro selection experiments o-phosphate of a 5’-triphosphate [4,5]. Some of these involving iterative cycles of affinity chromatography and ribozymes may be suitable starting points for the ia vitro sequence amplification have led to the isolation of speci- evolution of ribozyme polymerases that use nucleoside fic RNAs that bind a wide range of small molecules with (or oligonucleotide) triphosphates as substrates. high specificity [2]. D’erect selections for catalytic RNAs from partially and completely random sequence RNA In order to isolate other ribozymes that might be pools have yielded several novel classes of ribozymes, evolved into replicases, we have started to explore the use including self-cleaving RNAs [3] and a variety of ligases of other phosphate activation chemistries. Nucleoside [4,5]. More complex ribozymes that bind two substrates phosphorimidazolides have been used extensively by and release two products have recently been isolated Orgel and colleagues [14,15] in studies of non-enzy- from pools of sequences biased towards a binding site for matic template-directed copying reactions, because they one substrate [6,7]. The chemistry catalyzed by ribo- are more highly activated than nucleoside triphosphates zymes has recently been extended beyond phospho- and therefore react more rapidly in uncatalyzed reac- transfer reactions with the isolation of a rotational tions. This higher uncatalyzed reaction rate led us to isomerase [8], a self-alkylating ribozyme [9] and ribo- hypothesize that it might be easier to isolate ribozymes zymes that carry out aminoacyl transfer reactions [lo] (P that would join phosphorimidazolide-derivatized oligo- Lohse and J.W. S., unpublished data). nucleotides instead of triphosphate-activated oligo- nucleotides, because a smaller rate enhancement would RNA can catalyze several reactions that lead to polynu- still provide a useful absolute reaction rate. Here we cleotide synthesis [l 1,121.We have found that derivatives present the results of an attempt to use in vitro selection of group I introns are able to join together a series of to isolate novel ribozymes that ligate their own 3’ ends short oligonucleotides that have been aligned on a tem- to a 5’-phosphorimidazolide-activated oligonucleotide plate strand, producing a complementary strand [13]. substrate. Instead of the desired activity, we found However, the transesterification reaction catalyzed by ribozymes that ligated their 5’ ends to the substrate. *Corresponding author. +Present address: Geron Corporation, 200 Constitution Drive, Menlo Park, CA 94025, USA. 0 Current Biology Ltd ISSN ;074-5521 325 326 Chemistry & Biology 1995, Vol 2 No 5 Fig. 1. In vitro selection scheme for iso- (a) lating RNAs that self-ligate to a short substrate oligonucleotide. (a) The RNA 0 8 f; 5’ PBS pool contains 90 nucleotides of random II 20 bases -o- p-o- p-o- y-0 sequences flanked by defined primer I Random RNA sequences binding sites (PBS) for PCR amplifica- -0 -0 -0 tion. The substrate is a six-nucleotide synthetic RNA, which is biotinylated at its 3’ end and activated with a 5’-phos- 04 phorimidazolide. The 3’-PBS of the RNA pool contains a sequence complemen- tary to the substrate oligonucleotide, F; designed so that binding of the substrate -o_~-o-~-o_p_o oligonucleotide would form an inter- I I 5’ PBS 3’ PBS \ -0 -0 -0 rupted hairpin-loop, which would juxta- ’ pose the 3’.OH of the pool RNA with the 5’-phosphorimidazole moiety of the substrate. (b) The reaction catalyzed by the RNA that was actually selected from the pool: attack of the y-phosphate of the 5’-triphosphate of the pool RNA on the 5’-phosphorimidazolide of the sub- strate to generate a 5’4 tetraphosphate 0 0 Bintin.! II II FI f linkage. Molecules that became ligated o-~-o-_8-o-P-o-P-o 3’ PBS to the substrate were purified away from Oligo 5’ PBS substrate -O -0 -0 -0 unligated RNAs by binding to strept- avidin agarose. These RNAs were eluted, reverse transcribed, PCR ampli- fied and transcribed with T7 RNA poly- merase, generating more RNA for further rounds of selection. Bind to streptavidin agarose column Wash extensively Elute bound RNAs t Amplify Results The 3’ primer binding site was designed to fold into a In vitro selection partial hairpin stem-loop adjacent to the substrate- An in vitro selection experiment involves the synthesis of binding site such that Watson-Crick base pairing with a suitable pool of random-sequence RNA molecules, the substrate hexanucleotide would position the followed by the incubation of the pool with a substrate 3’-hydroxyl of the pool RNA to attack the 5’-phosphor- and the subsequent selection from the pool of those rare imidazolide of the activated substrate (Fig. la).The tem- molecules that are able to modify themselves by reacting plate-directed but otherwise uncatalyzed condensation of with the substrate. In the selection described here, the imidazolide-activated oligonucleotides generates pre- substrate was a hexanucleotide with a 5’-phosphate that dominantly 3’-5’ phosphodiester linkages (R. Rohatgi was activated by reaction with the water-soluble carbo- and J.WS., unpublished data). The rate of ligation of diimide EDC and imidazole to form the reactive random pool RNA with saturating concentrations of this phosphorimidazolide (Fig. 1). The substrate was also substrate was -7 x lop6 min-I, while reaction with a dif- biotinylated at its 3’ end, allowing the reacted molecules ferent substrate, not complementary to the template to be purified using streptavidin agarose. region, was undetectable after a 24 h reaction. A random-sequence RNA pool was constructed by in In the first round of in vitro selection, the activated sub- vitro transcription of a synthetic oligodeoxynucleotide strate was incubated with the random-sequence RNA that had been amplified by PCR.The pool had a com- for 4 h in a buffer containing high concentrations of salt plexity of -1 x 1015 different molecules and each RNA and magnesium (to stabilize folded RNA structures). molecule contained 90 random nucleotides flanked by Pool molecules that had become covalently linked to defined primer-binding sites of 20 and 39 nucleotides. the biotinylated substrate were purified by streptavidin A 5’-5’ ligase ribozyme Chapman and Szostak 327 Table 1. Selection Summary Round Amount of RNA Reaction time Mg2+ cont. % recovered 1 1 Tz 4h 50 mM 0.03 % 2 50 Pg 45 min 50 mM 1.5 % 3 50 Pg 30 min 50 mM 0.008 % 4 50 r*g 30 min 50 mM 0.024 % 5 50 pg 30 min 50 mM 0.02 % 6 50 kg 20 min 50 mM 2.0 % 7 50 )Lg 2 min 50 mM 0.7 % 8 50 tJg 2 min 100 FM 0.03 % Summary of reaction conditions throughout the selection. Percentage recovered is percentage of input RNA recovered from the streptavidin column. Following round 2, the column washihg conditions were altered to include a denaturing wash to reduce non- specific sticking to the column. affinity chromatography, reverse transcribed and PCR The secondary structure of the 54-nucleotide deletion amplified (Fig.
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