Flexizymes: Their Evolutionary History and the Origin of Catalytic Function

Abstract

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino...

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

“…Alternatively, non-natural amino acids ( N -methylated amino acids or unnatural side chains) (Josephson et al , 2005; Goto and Suga, 2012; Hipolito and Suga, 2012), can in principle be incorporated via the BeSD, e.g. when flexizyme (Morimoto et al , 2011) is added to activate a range of non-natural amino acids for in vitro incorporation.…”

Methods for the Creation of Cyclic Peptide Libraries for Use in Lead Discovery

“…45 Although not suitable for the intracellular production of cyclic peptides, flexizymes have been extensively employed for the in vitro production of cyclic peptide libraries that contain nonnatural amino acids. Flexizymes have been most widely combined with the PURE in vitro transcription/translation (IVTT) system.…”

Genetically Encoded Libraries of Nonstandard Peptides

“…The prototype [102] of the flexizyme used in genetic code reprogramming, which was evolved from a random RNA pool, has expanded its acceptability of tRNAs [103, 104] and amino acids for aminoacylation and turned into highly flexible ribozymes for tRNA aminoacylation [101, 105, 106]. Because the flexizymes recognize their cognate aromatic group, they are able to charge a wide variety of not only L-amino acids with nonproteinogenic side chains [3, 107–111], but also amino acids with an altered backbone such as N -alkyl amino acids [112–114], N -acyl amino acids [115], D-amino acids [116], β -amino acids, oligopeptides [117, 118], and even hydroxy acids [119, 120].…”