Non-Watson-Crick RNA synthesis suited to origin functions.

A templated RNA synthesis is characterized in which GppG accelerates synthesis of AppA from pA and chemically activated ImpA precursors. Similar acceleration is not observable in the presence of UppU, CppC, AppG, AppA, or pG alone. Thus, it seems likely that AppA is templated by GppG via a form or forms of G:A base-pairing.

Cross-backbone templating; ribodinucleotides made on poly(C).

G(5')pp(5')G synthesis from pG and chemically activated 2MeImpG is accelerated by the addition of complementary poly(C), but affected only slightly by poly(G) and not at all by poly(U) and poly(A). This suggests that 3'-5' poly(C) is a template for uncatalyzed synthesis of 5'-5' GppG, as was poly(U) for AppA synthesis, previously. The reaction occurs at 50 mM mono- and divalent ion concentrations, at moderate temperatures, and near pH 7.

Poly(U) RNA-templated synthesis of AppA.

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5'-5' AppA synthesis by 3'-5' poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis.

The plausibility of RNA-templated peptides: simultaneous RNA affinity for adjacent peptide side chains.

According to the RNA world hypothesis, coded peptide synthesis (translation) must have been first catalyzed by RNAs. Here, we show that small RNA sequences can simultaneously bind the dissimilar amino acids His and Phe in peptide linkage. We used in vitro counterselection/selection to isolate a pool of RNAs that bind the dipeptide NH(2)-His-Phe-COOH with K (D) ranging from 36 to 480 μM.

Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA.

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved.

A diminutive and specific RNA binding site for L-tryptophan.

Selection for amino acid affinity by elution of RNAs from tryptophan-Sepharose using free L-tryptophan evokes one sequence predominantly (K(D) = 12 microM), a symmetrical internal loop of 3 nt per side. Though we have also isolated larger sequences with affinity for tryptophan, successively squeezed selection in randomized tracts of 70, 60, 40, 20 and 17 nt show that this internal loop is the simplest sequence that can meet the column affinity selection. From sequence variation in approximately 50 independent isolates, only 26 bits of information are required to describe this loop (equivalent to only 13 fully conserved nucleotides).

RNA affinity for molecular L-histidine; genetic code origins.

Selection for affinity for free histidine yields a single RNA aptamer, which was isolated 54 times independently. This RNA is highly specific for the side chain and binds protonated L-histidine with 10(2)-10(3)-fold stereoselectivity and a dissociation constant (K(D)) of 8-54 microM in different isolates. These histidine-binding RNAs have a common internal loop-hairpin loop structure, based on a conserved RAAGUGGGKKN(0-36) AUGUN(0-2)AGKAACAG sequence.

Selection of the simplest RNA that binds isoleucine.

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides.