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NASA NTRS2023

Peptide conjugates with polyaromatic hydrocarbons can benefit the activity of catalytic RNAs

Early stages of life likely employed catalytic RNAs (ribozymes) in many functions that are today filled by proteins. However, the earliest life forms must have emerged from heterogenous chemical mixtures, which included amino acids, short peptides, and many other compounds. Here we explored whether the presence of short peptides can help the emergence of catalytic RNAs. To do this, we conducted an in vitro selection for catalytic RNAs from randomized sequence in the presence of ten different peptides with a prebiotically plausible length of eight amino acids. This in vitro selection generated dozens of ribozymes, one of them with ∼900-fold higher activity in the presence of one specific peptide. Unexpectedly, the beneficial peptide had retained its N-terminal Fmoc protection group, and this group was required to benefit ribozyme activity. The same, or higher benefit resulted from peptide conjugates with prebiotically plausible polyaromatic hydrocarbons (PAHs) such as fluorene and naphthalene. This shows that PAH-peptide conjugates can act as potent cofactors to enhance ribozyme activity. The results are discussed in the context of the origin of life.

Kevin J. Sweeney↗

NASA NTRS2003

Catalytic Activity of a Binary Informational Macromolecule

RNA molecules are thought to have played a prominent role in the early history of life on Earth based on their ability both to encode genetic information and to exhibit catalytic function. The modern genetic alphabet relies on two sets of complementary base pairs to store genetic information. However, due to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish. It has been suggested that the first genetic material instead contained only a single base-pairing unie'. Here we show that binary informational macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain ligase ribozymes composed of only 2,6-diaminopurine and uracil nucleotides, which catalyze the template-directed joining of two RNA molecules, one bearing a 5'-triphosphate and the other a 3'-hydroxyl. The active conformation of the fastest isolated ribozyme had a catalytic rate that was about 36,000-fold faster than the uncatalyzed rate of reaction. This ribozyme is specific for the formation of biologically relevant 3',5'-phosphodiester linkages.

Reader, John S.↗

NASA NTRS1995

Structurally complex and highly active RNA ligases derived from random RNA sequences

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Non-NASA Center↗

NASA NTRS2017

Exploring Connectivity in Sequence Space of Functional RNA

Emergence of replicable genetic molecules was one of the marking points in the origin of life, evolution of which can be conceptualized as a walk through the space of all possible sequences. A theoretical concept of fitness landscape helps to understand evolutionary processes through assigning a value of fitness to each genotype. Then, evolution of a phenotype is viewed as a series of consecutive, single-point mutations. Natural selection biases evolution toward peaks of high fitness and away from valleys of low fitness. whereas neutral drift occurs in the sequence space without direction as mutations are introduced at random. Large networks of neutral or near-neutral mutations on a fitness landscape, especially for sufficiently long genomes, are possible or even inevitable. Their detection in experiments, however, has been elusive. Although a few near-neutral evolutionary pathways have been found, recent experimental evidence indicates landscapes consist of largely isolated islands. The generality of these results, however, is not clear, as the genome length or the fraction of functional molecules in the genotypic space might have been insufficient for the emergence of large, neutral networks. Thorough investigation on the structure of the fitness landscape is essential to understand the mechanisms of evolution of early genomes. RNA molecules are commonly assumed to play the pivotal role in the origin of genetic systems. They are widely believed to be early, if not the earliest, genetic and catalytic molecules, with abundant biochemical activities as aptamers and ribozymes, i.e. RNA molecules capable, respectively, to bind small molecules or catalyze chemical reactions. Here, we present results of our recent studies on the structure of the sequence space of RNA ligase ribozymes selected through in vitro evolution. Several hundred thousands of sequences active to a different degree were obtained by way of deep sequencing. Analysis of these sequences revealed several large clusters defined such that every sequence in a cluster can be reached from any other sequence in the same cluster through a series of single point mutations. Sequences in a single cluster appear to adopt more than one secondary structure. The mechanism of refolding within a single cluster was examined. To shed light on possible evolutionary paths in the space of ribozymes, the connectivity between clusters was investigated. The effect of length of RNA molecules on the structure of the fitness landscape and possible evolutionary paths was examined by way of comparing functional sequences of 20 and 80 nucleobases in length. It was found that sequences of different lengths shared secondary structure motifs that were presumed responsible for catalytic activity, with increasing complexity and global structural rearrangements emerging in longer molecules.

Wei, Chenyu↗

NASA NTRS2017

Analysis of in Vitro Evolution Reveals the Underlying Distribution of Catalytic Activity Among Random Sequences

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher’s Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

Abe Pressman↗

NASA NTRS2001

Hydrolysis of tRNA(sup Phe) on Suspensions of Amino Acids

RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNA(sup Phe) is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and beta-glutamic acid but not by suspensions of alpha-glutamic acid, asparagine, or glutamine. The non-enzymatic hydrolysis of RNA has been studied extensively, especially because of its relevance to the mechanisms of action of ribozymes and to biotechnology and therapy. Many ribonucleases, ribozymes, and non-biological catalysts function via acid-base catalysis of an intramolecular transesterification mechanism in which the 2'-OH group attacks the adjacent phosphate group. The pentacoordinated phosphorane intermediate may collapse back to starting material, or yield isomerized or cleaved products.

Gao, Kui↗

NASA NTRS1994

In vitro selection of catalytic RNAs

In vitro selection techniques are poised to allow a rapid expansion of the study of catalysis by RNA enzymes (ribozymes). This truly molecular version of genetics has already been applied to the study of the structures of known ribozymes and to the tailoring of their catalytic activity to meet specific requirements of substrate specificity or reaction conditions. During the past year, in vitro selection has been successfully used to isolate novel RNA catalysts from random sequence pools.

NASA Discipline Exobiology↗

NASA NTRS1999

In vitro selection of functional nucleic acids

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

Review↗

NASA NTRS1999

Non-unity molecular heritability demonstrated by continuous evolution in vitro

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

Non-NASA Center↗

NASA NTRSDate not supplied

In Vitro Selections with RNAs of Variable Length Converge on a Robust Catalytic Core

In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity. We identified a core ribozyme structure that was robust to changes in RNA length, high levels of mutagenesis, and increased selection pressure. Elaboration on this core structure resulted in improved activity which we show is consistent with a larger trend among functional RNAs in which increasing motif size can lead to an exponential improvement in fitness. We conclude that elaboration on conserved core structures is a preferred mechanism in RNA evolution. This conclusion, drawn from selections of RNAs from random sequences, is consistent with proposed evolutionary histories of specific biological RNAs. More generally, our results indicate that modern RNA structures can be used to infer ancestral structures. Our observations also suggest a mechanism by which structural outcomes of early RNA evolution would be largely reproducible even though RNA fitness landscapes consist of disconnected clusters of functional sequences.

In vitro evolution↗

NASA NTRS1987

The case for an ancestral genetic system involving simple analogues of the nucleotides

The idea that the first living systems on earth were based on self-replicating RNA molecules has recently become popular as a result of the discovery of ribozymes. However, there are several major problems associated with the prebiotic synthesis of ribonucleotides. In addition, there is the newly recognized problem of enantiomeric cross-inhibition, whereby template-directed polymerization involving one enantiomer of RNA is inhibited strongly by the presence of the other enantiomer. Here, it is proposed that RNA was preceded in the evolution of life by a polymer constructed from flexible, acyclic, probably prochiral nucleotide analogues that were synthesized readily on the primitive earth. Several potentially prebiotic nucleotide analogues are considered in this context, and some of the consequences of this proposal are discussed.

Joyce, Gerald F.↗

NASA NTRS1991

Macromolecular recognition: Structural aspects of the origin of the genetic system

Theoretical simulation of prebiotic chemical processes is an invaluable tool for probing the phenomenon of evolution of life. Using computational and modeling techniques and guided by analogies from present day systems we, seek to understand the emergence of genetic apparatus, enzymatic catalysis and protein synthesis under prebiotic conditions. In one possible scenario, the RNA enzymatic reaction plays a key role in the emergence of the self-replicating and offers a clue to the onset of enzymatic catalysis prior to the existence of the protein biosynthetic machinery. Our ultimate goal is to propose a simple RNA segment which contains the specificity and catalytic activity of the contemporary RNA enzyme and which could emerge in a primordial chemical environment. To understand the mechanism of ribozyme catalyzed reactions, ab initio and semi-empirical (ZINDO) programs were used to investigate the reaction path of transphosphorylation. A special emphasis was placed on the possible catalytic and structural roles played by the coordinated magnesium cation. Both the inline and adjacent mechanisms of transphosphorylation have been studied. Another important aspect of this reaction is the identity of the functional groups which are essential for the acid base catalysis. The structural characteristics of the target helices, particularly a possible role of G center dot T pair, is under examination by molecular dynamics (MD) simulation technique. Modeling of the ancestral aminoacyl-tRNA synthetases (aRS) may provide important clues to the emergence of the genetic code and the protein synthetic machinery. Assuming that the catalytic function evolved before the elements of specific recognition of a particular amino acid, we are exploring the minimal structural requirements for the catalysis of tRNA aminoacylation. The molecular modeling system SYBYL was used for this study based on the high resolution crystallographic structures of the present day tyrosyl-adenylate:tyrRS and tRNA(Gln): ATP:glnRS complexes. The trinucleotide CCA of the 3'-end tRNA is placed into the active site pocket of tyrRS, based on the scheme of interaction between tRNA(Gln) and glnRS, and upon the stereochemistry of the tyrRS:tRNA:Tyr-AMP transition state. This provides a model of the non-specific recognition of a tRNA's 3'-end by an aRS, which might be similar to that of the ancestral aRS's. In the next step, modeling of the rest of the acceptor stem of tRNA (Tyr) with tyrRS is carried out.

Rein, Robert↗

NASA NTRS1991

Catalytic RNA and synthesis of the peptide bond

We are studying whether the L-19 IVS ribozyme from Tetrahymena thermophila can catalyze the formation of the peptide bond when it is supplied with synthetic aminoacyl oligonucleotides. If this reaction works, it could give us some insight into the mechanism of peptide bond formation and the origin of coded protein synthesis. Two short oligoribonucleotides, CCCCC and a protected form of CCCCU were prepared; the former was made by the controlled hydrolysis of Poly(C), and the later by multistep chemical synthesis from the protected monomers. The homopentamer was then aminocylated using C-14 labelled Boc-protected glycine imidazolide. This aminoacylated oligo-nucleotide has now been shown to enter the active site of the L-19 IVS, and aminoacyl transfer, and peptide bond formation reactions are being sought. Our synthesis of CCCCU made us aware of the inadequacy of many of the 2'- hydroxyl protecting groups that are in use today and we therefore designed a new 2'- protecting group that is presently being tested.

Usher, D. A.↗

NASA NTRS1991

Macromolecular recognition: Structural aspects of the origin of the genetic system

Theoretical simulation of prebiotic chemical processes is an invaluable tool for probing the phenomenon of the evolution of life. Using computational and modeling techniques and guided by analogies from present day systems, we seek to understand the emergence of the genetic apparatus, enzymatic catalysis and protein synthesis under prebiotic conditions. Modeling of the ancestral aminoacyl-tRNA-synthetases (aRS) may provide important clues to the emergence of the genetic code and the protein synthetic machinery. The minimal structural requirements for the catalysis of tRNA aminoacylation are being explored. A formation of an aminoacyl adenylate was studied in the framework of ab initio molecular orbital theory. The role of individual residues in the vicinity of the TyrRS active site was examined, and the effect of all possible amino acids substitutions near the active site was examined. A formation of aminoacyl tRNA was studied by the molecular modeling system SYBYL with the high resolution crystallographic structures of the present day tRNA, aRS's complexes. The ultimate goal is to propose a simple RNA segment that is small enough to be build in the primordial chemical environment but maintains the specificity and catalytic activity of the contemporary RNA enzyme. To understand the mechanism of ribozyme catalyzed reactions, ab initio and semi-empirical (ZINDO) programs were used to investigate the reaction path of transphosphorylation. A special emphasis was placed on the possible catalytic and structural roles played by the coordinated magnesium cation. Both the inline and adjacent mechanisms of transphosphorylation were studied. The structural characteristics of the target helices, particularly a possible role for the G-T pair, is also studied by a molecular dynamics (MD) simulation technique.

Rein, Robert↗

NASA NTRS1994

Template-directed chemistry and the origins of the RNA world

Prompted by the growing number of reports about reactions catalyzed by ribozymes, this paper summarizes mechanistic and kinetic aspects of template-directed (TD) chemistry important for the synthesis of a diverse population of polynucleotides and analogues possibly up to 100 units long. Assuming that this chemistry takes place in a microenvironment conducive to life under the constant influx of mM concentrations of activated monomeric building blocks, the proposed scenario represents a working hypothesis for the prebiotic synthesis of the RNA world.

Kanavarioti, Anastassia↗

NASA NTRS1993

The RNA-world and co-evolution hypothesis and the origin of life: Implications, research strategies and perspectives

The applicability of the RNA-world and co-evolution hypothesis to the study of the very first stages of the origin of life is discussed. The discussion focuses on the basic differences between the two hypotheses and their implications, with regard to the reconstruction methodology, ribosome emergence, balance between ribozymes and protein enzymes, and their major difficultites. Additional complexities of the two hypotheses, such as membranes and the energy source of the first reactions, are not treated in the present work. A central element in the proposed experimental strategies is the study of the catalytic activites of very small peptides and RNA-like oligomers, according to existing, as well as to yet-to-be-invented scenarios of the two hypothesis under consideration. It is suggested that the novel directed molecular evolution technology, and molecular computational modeling, can be applied to this research. This strategy is assumed to be essential for the suggested goal of future studies of the origin of life, namely, the establishment of a `Primordial Darwinian entity'.

Lahav, Noam↗

NASA NTRS2000

Remote Enantioselection Transmitted by an Achiral Peptide Nucleic Acid Backbone

short homochiral segment of DNA into a PNA helix could have guaranteed that the next short segment of DNA to be incorporated would have the same handedness as the first. Once two segments of the same handedness were present, the probability that a third segment would have the same handedness would increase, and so on. Evolution could then slowly dilute out the PNA part. This scenario would ultimately allow the formation of a chiral oligonucleotide by processes that are largely resistant to enantiomeric crossinhibition. It is important to note that the ligation of homochiral dinucleotides on a nucleic acid template would probably be at least as enantiospecific as the reaction that we have studied. The disadvantage of using chiral monomers as components of a replicating system arises from the difficulty of generating a first long homochiral template from a racemic mixture of monomers, although results of experiments designed to overcome this difficulty by employing homochiral tetramers have been reported.l l The probability of obtaining a homochiral n-mer from achiral substrates is approximately 1P-I if the nontemplate-directed extension of the primer is not enantioselective. Hence, it would be very hard to get started with a homochiral 40-mer, for example. No such difficulty exists in a scenario that originates with an achiral genetic material and in which the incorporation of very few chiral monomers in this achiral background gradually progresses towards homochirality. It seems possible that some PNA sequences could act as catalysts, analogous to ribozymes, even though PNA lacks clear metal binding sites. Although such catalysts could not be enantioselective, the incorporation of as few as two chiral nucleotides could then impose chiral specificity on the system. Furthermore, such patch chimeras could help to bridge the gap in catalytic potential between PNA and RNA, while guaranteeing enantioselectivity.

Kozlov, Igor A.↗

NASA NTRS1988

A molecular orbital study of a model of the Mg2+ coordination complex of the self splicing reaction of ribosomal RNA

Recent discoveries have established the fact that RNA is capable of acting as an enzyme. In this study two different types of molecular orbital calculations, INDO and ab initio, were used in an attempt to assess the structural/functional role of the Mg2+ hydrated complex in ribozyme reactions. Preliminary studies indicate that the reaction is multistep and that the Mg2+ complex exerts a stabilizing effect on the intermediate or midpoint of the reaction.

Non-NASA Center↗