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Project Progress

Thanks to earlier experiments within this project, as well as related work in my and other laboratories, it is accepted by a majority of evolutionarily concerned biologists that our immediate predecessors were a biota that made more extensive use of a molecule very like RNA, perhaps using it as the only macromolecule for a time. This congener of RNA thereby predates other modern macromolecules and more recently (post RNA world) ceded many cellular functions to the other characters of the central dogma, DNA and protein. One influential consequence of these views is that RNA must have devised translation (so that RNA catalysts could be replaced with peptides), and therefore RNA must be competent in all reactions required for coded peptide synthesis. We have published an experimental demonstration that this is a biochemically plausible proposition by showing that all types of reactions required for translation are within the RNA repertoire (1).

Thus it is likely that amino acids were admitted to proteins by the action of RNA. This immediately raises the possibility that the universal use of L-amino acids in proteins may have occurred because these amino acids were chosen in interactions with the chiral reagent RNA, with its D-ribose backbone. This, even if true, would not solve the problem of biological asymmetry, but would displace the biological breaking of chirality to the origin of RNA and therefore represent progress on one of the 'great questions’ of molecular evolution.

This idea has been pursued in the past by measuring differential rates of aminoacylation of ribonucleotide models by activated D and L-amino acids. Profy and Usher (2) used racemic DNB-alanine imidazolides to acylate poly A, I, C and U. They found L was added more rapidly from almost 4-fold (poly A) to about 2-fold (poly C). However, when the DNB protecting group was removed, more nearly emulating aa-tRNA synthesis, the stereospecificity flipped, with D-amino acids selected by D-RNA. Tamura and Schimmel (3) more recently used a specific molecular model consisting of a 5’ p-amino acid (emulating an amino acid adenylate) at a nick in an RNA-hybrid helix. The activated L-alanine was transferred across the nick to adenosine 3’ OH at 4 times the rate of D-alanine.

Thus there is an indication of moderate stereospecificity in the literature. However, the results may not generalize given that a modest quantitative outcome depended on the sequence of the RNA, and that only alanine has been used. Furthermore, unknown constraints are hidden in both experiments in that rates (and therefore incompletely known transition states) are being surveyed, and in the Tamura-Schimmel case the effect of an arbitrarily chosen helix geometry may possibly be influential.

It might therefore be useful to ask in a more open-ended way if there is a chiral interaction between L-amino acids and D-RNAs. The basic idea is to give RNAs an unbiased chance to bin D- & L-amino acids, and to ask how well, relatively, they can do this. Our usual affinity selection protocols are well suited to this question, so we have tried to adapt them to determine chiral preference.

The basic idea is to do a selection with equal amounts of D- and L-amino acids immobilized in a column exposed to 10¹⁴ to 10¹⁵ different RNA sequences. The column is eluted with a racemic mix of amino acid, and the RNA re-amplified until RNA that binds is isolated. Under this even-handed selection, does RNA binding the L- or the D-amino acid arise first? This protocol is understood to some extent, and is a plausible way to ask whether the most abundant sequences that bind an amino acid are stereoselective (conceivably they might be unselective).

The most interesting example of this experiment we have carried out (related selection in 4) relies on selection for binding DL-histidine columns and elution by racemic histidine. One requirement for a clear experiment is that there be no other chiral molecules in the selection, so that observed chiral preferences can be attributed unambiguously to a choice between the amino acids. This makes our normal column procedure unusable because we use Sepharose, a natural product made of chiral sugars. Therefore we first sought a new non-chiral column material (but see below for more on Sepharose).

This proved to be a considerable obstacle, as the obvious alternatives were all worse; we eliminated polyacrylamide, latex and polystyrene supports on various grounds, such as non-specific affinity for RNA, incompatibility with the solvents needed to derivatize with amino acids, and the like. We finally ended up with controlled pore glass (CPG), available with long support arms terminated in an amine. This meets the theoretical requirement nicely, because glass is a frozen liquid and can have no underlying chiral bias. D-his-glass and L-his glass were mixed in equal quantities to make columns in which the two forms of the amino acid must have equal abundances.

In brief, the selection with DL-his-glass (bound through the carboxyl group) gave an extremely clear result. The first RNA to elute with histidine was entirely L-specific, whether tested as a pool or resolved into separate sequenced isolates and tested as pure single RNA transcripts. The L-histidine binding RNA consisted of a single species of aptamer that must have been one or two orders of magnitude more abundant than D-histidine aptamers.

An important control was carried out: when the selection was carried out again from two cycles earlier, but eluting with D-histidine only, RNAs that eluted with the D-amino acid were easily recovered. This shows that the techniques were sufficient to recover the other specificity and indeed, that such RNAs existed in our experiment. Thus the predominance of the L-his aptamers was not due to some cryptic defect in the selection for D-his affinity. Given these results, it appears that L-preference may be generalizable. This would be an exciting evolutionary result, arguing that the universal handedness of modern proteins is due to RNA activity. However, now our old favorite Sepharose appears again: as a warm up using known techniques, we had previously selected on DL-phenylalanine-Sepharose using the same protocols. The results were as definitive in favor of D-phe as they had been for L-his on CPG! So now we have a dilemma — are the results different for different amino acid sidechains (thereby giving us a clue about which amino acids entered biology via RNA activity) or is this difference the anticipated confusion arising from a chiral support? Stay tuned.

References

1. Yarus M. 2001. Cold Spring Harb Symp Quant Biol 66: 207-15
2. Profy AT, Usher DA. 1984. J Am Chem Soc 106: 5030-31
3. Tamura K, Schimmel P. 2004. Science 305: 1253.
4. Majerfeld, I, Puthenvedu, D, Yarus, M. 2005. J Mol Evol , RNA Affinity for Molecular L-Histidine; Genetic Code Origins, online ahead of print, PubMed PMID: 15999244