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1.  Getting Past the RNA World: The Initial Darwinian Ancestor 
A little-noted result of the confirmation of multiple premises of the RNA-world hypothesis is that we now know something about the dawn organisms that followed the origin of life, perhaps over 4 billion years ago. We are therefore in an improved position to reason about the biota just before RNA times, during the era of the first replicators, the first Darwinian creatures on Earth. An RNA congener still prominent in modern biology is a plausible descendent of these first replicators.
Nucleotide cofactors such as NADP could represent relics of pre-RNA life on Earth. They may have been key elements of the first replicators, subsequently co-opted by RNA and then proteins.
PMCID: PMC3062219  PMID: 20719875
2.  A ribonucleotide Origin for Life – Fluctuation and Near-ideal Reactions 
Oligoribonucleotides are potentially capable of Darwinian evolution – they may replicate and can express an independent chemical phenotype, as embodied in modern enzymatic cofactors. Using quantitative chemical kinetics on a sporadically fed ribonucleotide pool, unreliable supplies of unstable activated ribonucleotides A and B at low concentrations recurrently yield a replicating AB polymer with a potential chemical phenotype. Self-complementary replication in the pool occurs during a minority (here ≈ 35 %) of synthetic episodes that exploit coincidental overlaps between 4, 5 or 6 spikes of arbitrarily arriving substrates. Such uniquely productive synthetic episodes, in which near-ideal reaction sequences recur at random, account for most AB oligonucleotide synthesis, and therefore underlie the emergence of net replication under realistic primordial conditions. Because overlapping substrate spikes are unexpectedly frequent, and in addition, complex spike sequences appear disproportionately, a sporadically fed pool can host unexpectedly complex syntheses. Thus, primordial substrate fluctuations are not necessarily a barrier to Darwinism, but instead can facilitate early evolution.
PMCID: PMC3576565  PMID: 23344886
Cofactor; Origin of life; Replication; Selection; Initial Darwinian Ancestor
3.  Small aminoacyl transfer centers at GU within a larger RNA 
RNA Biology  2012;9(1):59-66.
Separate aminoacyl transfer centers related to the small …GUNNN..: NNNU ribozyme seem possible at the frequent GU sequences dispersed throughout an RNA tertiary structure. In fact, such activity is easily detected and varies more than 2 orders in rate, probabably being faster at sites with less structural constraint. Analysis of a particular constrained active site in an rRNA transcript suggests that its difficulty lies not in substrate strand association, but in binding and/or group transfer from the aminoacyl precursor. Efficient aminoacyl transfer requires accurate complementarity between large or small ribozymes and oligoribonucleotide substrates, even when only three or four base pairs link the two. Thus, multi-site active ribozymal superstructures might have coordinated an RNA metabolism, including aiding an early translation apparatus.
PMCID: PMC3342943  PMID: 22258149
adenylate; aminoacylation; enzyme; oligonucleotide; rRNA
4.  The meaning of a minuscule ribozyme 
The smallest ribozyme that carries out a complex group transfer is the sequence GUGGC-3′, acting to aminoacylate GCCU-3′ (and host a manifold of further reactions) in the presence of substrate PheAMP. Here, I describe the enzymatic rate, the characterization of about 20 aminoacyl-RNA and peptidyl-RNA products and the pathways of these GUGGC/GCCU reactions. Finally, the topic is evolution, and the potential implications of these data for the advent of translation itself.
PMCID: PMC3158920  PMID: 21930581
RNA; aminoacylation; aminoacyl-RNA; peptidyl-RNA; translation
5.  Darwinian Behavior in a Cold, Sporadically Fed Pool of Ribonucleotides 
Astrobiology  2012;12(9):870-883.
A testable, explicit origin for Darwinian behavior, feasible on a chaotic early Earth, would aid origins discussion. Here I show that a pool receiving unreliable supplies of unstable ribonucleotide precursors can recurrently fill this role. By using numerical integration, the differential equations governing a sporadically fed pool are solved, yielding quantitative constraints for the proliferation of molecules that also have a chemical phenotype. For example, templated triphosphate nucleotide joining is >104 too slow, suggesting that a group more reactive than pyrophosphate activated primordial nucleotides. However, measured literature rates are sufficient if the Initial Darwinian Ancestor (IDA) resembles a 5′-5′ cofactor-like dinucleotide RNA, synthesized via activation with a phosphorimidazolide-like group. A sporadically fed pool offers unforeseen advantages; for example, the pool hosts a novel replicator which is predominantly unpaired, even though it replicates. Such free template is optimized for effective selection during its replication. Pool nucleotides are also subject to a broadly based selection that impels the population toward replication, effective selection, and Darwinian behavior. Such a primordial pool may have left detectable modern traces. A sporadically fed ribonucleotide pool also fits a recognizable early Earth environment, has recognizable modern descendants, and suits the early shape of the phylogenetic tree of Earthly life. Finally, analysis points to particular data now needed to refine the hypothesis. Accordingly, a kinetically explicit chemical hypothesis for a terran IDA can be justified, and informative experiments seem readily accessible. Key Words: Cofactor—RNA—Origin of life—Replication—Initial Darwinian Ancestor (IDA). Astrobiology 12, 870–883.
PMCID: PMC3444769  PMID: 22946838
6.  The Plausibility of RNA-Templated Peptides: Simultaneous RNA Affinity for Adjacent Peptide Side Chains 
Journal of Molecular Evolution  2012;74(3-4):217-225.
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 NH2-His-Phe-COOH with KD ranging from 36 to 480 μM. These sites contact both side chains, usually including the protonated imidazole of His, but bind-free l-His and l-Phe with much lower, sometimes undetectable, affinities. The most frequent His–Phe sites do not usually contain previously isolated sites for individual amino acids, and are only ≈35 % larger than previously known separate His and Phe sites. Nonetheless, His–Phe sites appear enriched in His anticodons, as previous l-His sites also were. Accordingly, these data add to existing experimental evidence for a stereochemical genetic code. In these peptide sites, bound amino acids approach each other to a proximity that allows a covalent peptide linkage. Isolation of several RNAs embracing two amino acids with a linking peptide bond supports the idea that a direct-RNA-template could encode primordial peptides, though crucial experiments remain.
Electronic supplementary material
The online version of this article (doi:10.1007/s00239-012-9501-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3346935  PMID: 22538927
Aptamer; Binding; Histidine; Phenylalanine; Genetic code; Life Sciences; Animal Genetics and Genomics; Cell Biology; Microbiology; Plant Sciences; Plant Genetics & Genomics; Evolutionary Biology
7.  Specific RNA binding to ordered phospholipid bilayers 
Nucleic Acids Research  2006;34(7):2128-2136.
We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.
PMCID: PMC1449910  PMID: 16641318
8.  Abundance of correctly folded RNA motifs in sequence space, calculated on computational grids 
Nucleic Acids Research  2005;33(18):5924-5935.
Although functional RNA molecules are known to be biased in overall composition, the effects of background composition on the probability of finding a particular active site by chance has received little attention. The probability of finding a particular motif has important implications both for understanding the distribution of functional RNAs in ancient and modern organisms with varying genome compositions and for tuning SELEX pools to optimize the chance of finding specific functions. Here we develop a new method for calculating the probability of finding a modular motif containing base-paired regions, and use a computational grid to fold several hundred million random RNA sequences containing the core elements of the isoleucine aptamer and the hammerhead ribozyme to estimate the probability that a sequence containing these structural elements will fold correctly when isolated from background sequences of different compositions. We find that the two motifs are most likely to be found in distinct regions of compositional space, and that the regions of greatest abundance are influenced by the probability of finding the conserved bases, finding the flanking helices, and folding, in that order of importance. Additionally, we can refine our estimates of the number of random sequences required for a 50% probability of finding an example of each site in unbiased random pools of length 100 to 4.1 × 109 for the isoleucine aptamer and 1.6 × 1010 for the hammerhead ribozyme. These figures are consistent with the facile recovery of these motifs from SELEX experiments.
PMCID: PMC1258168  PMID: 16237127
9.  A diminutive and specific RNA binding site for L-tryptophan 
Nucleic Acids Research  2005;33(17):5482-5493.
Selection for amino acid affinity by elution of RNAs from tryptophan–Sepharose using free L-tryptophan evokes one sequence predominantly (KD = 12 µM), 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 ∼50 independent isolates, only 26 bits of information are required to describe this loop (equivalent to only 13 fully conserved nucleotides). Thus, it is among the simplest amino acid binding sites known, as well as selective among hydrophobic side chains. Among site sequences defined as essential to affinity by conservation, protection and modification-interference, there is a recurring CCA sequence (a tryptophan anticodon triplet) which apparently forms one side of the binding site. Such conserved juxtaposition of tryptophan with a cognate coding triplet supports a stereochemical origin for the genetic code.
PMCID: PMC1236723  PMID: 16186130
10.  Analyzing partially randomized nucleic acid pools: straight dope on doping 
Nucleic Acids Research  2003;31(6):e30.
Partially randomized (doped) pools are important for optimizing activities initially isolated by selection-amplification or SELEX, and for locating nucleotides critical for function. Here we present a method for calculating the number of unique sequences in a pool, and the expected copy number of each unique sequence with a specified number of changes from the original sequence. Surprisingly, small differences in doping can have large consequences for the number of copies of sequences with certain numbers of changes from the original sequence. We demonstrate the effects of pool size, percentage doping, length of the random region and taking aliquots from the original pool on the exploration of sequence space in a doped reselection experiment. A web form is provided for customized calculations.
PMCID: PMC152884  PMID: 12626729
11.  The Process of Infection with Bacteriophage øX174 XIII. Evidence for an Essential Bacterial “Site” 1 
Journal of Virology  1967;1(1):135-144.
The burst of a starved bacterium infected with several øX174 bacteriophage was usually found to contain genetic traits of only one of the possible parents; less often, two phage multiplied in the same host cell. Unstarved cells, in contrast, supported the growth of at least four parental phage types. The unproductive phage seemed to be able to undergo the intracellular transition from parental single-stranded deoxyribonucleic acid to the double-stranded “replicative form” (RF). These results are taken to mean that some bacterial factor required for a step between RF synthesis and maturation of progeny is limited in starved cells.
PMCID: PMC375514  PMID: 4918231

Results 1-11 (11)