Engineering topics

Orgel, Leslie E.

Publications and source records attributed to Orgel, Leslie E..

At least 37 records · Page 2

NASA NTRS1994

In Vitro Selection of Optimal DNA Substrates for Ligation by a Water-Soluble Carbodiimide

We have used in vitro selection to investigate the sequence requirements for efficient template-directed ligation of oligonucleotides at 0 deg C using a water-soluble carbodiimide as condensing agent. We find that only 2 bp at each side of the ligation junction are needed. We also studied chemical ligation of substrate ensembles that we have previously selected as optimal by RNA ligase or by DNA ligase. As anticipated, we find that substrates selected with DNA ligase ligate efficiently with a chemical ligating agent, and vice versa. Substrates selected using RNA ligase are not ligated by the chemical condensing agent and vice versa. The implications of these results for prebiotic chemistry are discussed.

Harada, Kazuo↗

NASA NTRS1993

The limits of template-directed synthesis with nucleoside-5'-phosphoro(2-methyl)imidazolides

In earler work we have shown that C-rich templates containing isolated A, T or G residues and short oligo(G) sequences can be copied effectively using nucleoside-5'-phosphoro(2-methyl)imidazolides as substrates. We now show that isolated A or T residues within an oligo(G) sequence are a complete block to copying and that an isolated C residue is copied inefficiently. Replication is possible only if there are two complementary oligonucleotides each of which acts as a template to facilitate the synthesis of the other. We emphasize the severity of the problems that need to be overcome to make possible non-enzymatic replication in homogeneous aqueous solution. We conclude that an efficient catalyst was involved in the origin of polynucleotide replication.

Hill, Aubrey R.↗

NASA NTRS1993

In vitro selection of optimal DNA substrates for T4 RNA ligase

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

Harada, Kazuo↗

NASA NTRS1993

Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

Harada, Kazuo↗

NASA NTRS1993

The Limits of Template-Directed Synthesis with Nucleoside-5'-Phosphoro(2-Methyl) Imidazolides

In earlier work we have shown that C-rich templates containing isolated A, T or G residues and short oligo(G) sequences can be copied effectively using nucleoside-5'-phosphoro(2-methyl)imidazolides as substrates. We now show that isolated A or T residues within an oligo(G) sequence are a complete block to copying and that an isolated C residue is copied inefficiently. Replication is possible only if there are two complementary oligonucleotides each of which acts as a template to facilitate the synthesis of the other. We emphasize the severity of the problems that need to be overcome to make possible non-enzymatic replication in homogeneous aqueous solution. We conclude that an efficient catalyst was involved in the origin of polynucleotide replication.

Hill, Aubrey R., Jr.↗

NASA NTRS1992

Molecular replication

Recent experiments demonstrating nonenzymatic replication in molecular systems are reviewed. The difficulties facing nonenzymatic replication are discussed along with specificity, fidelity, and mutation in nonenzymatic replication. The prospects for research in this area are considered.

Orgel, Leslie E.↗

NASA NTRS1992

Nonenzymatic template-directed synthesis on hairpin oligonucleotides. II - Templates containing cytidine and guanosine residues

Hairpin oligonucleotides were prepared with 5-prime-terminal single-stranded sequments containing cytidylate (C) and guanylate (G) residues. It was found that incubation of these hairpin oligonucleotides with a mixutre of cytidine and guanosine 5-prime-phosphoro (2-methyl)imidazolides results in sequence-specific addition of C and G residues to the 3-prime terminus of the hairpin.

Wu, Taifeng↗

NASA NTRS1992

Nonenzymatic template-directed synthesis on hairpin oligonucleotides. III - Incorporation of adenosine and uridine residues

Nonenzymatic template-directed incorporation of adenosine and uridine residues into template sequences was obtained using nucleoside-5-prime phosphoro (2-methyl)imidazolides as substrates and hairpin oligonucleotides as templates. The reactions are regiospecific, producing mainly 3-prime-5-prime phosphodiester bonds. Limited synthesis of CA and AC sequences was observed along with some synthesis of the AA sequences on templates containing TG and GT sequences, along wilth some synthesis of the AA sequences on templates containing TT sequences.

Wu, Taifeng↗

NASA NTRS1991

Disulfide-linked oligonucleotide phosphorothioates - Novel analogues of nucleic acids

The synthesis of phosphorothioate analogs of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorotioate group undergoes efficient chain-extension when oxidized in the presence of 3.

Wu, Taifeng↗

NASA NTRS1991

The cyclization of arabinosyladenine-5-prime-phosphorimidazolide

When arabinosyladenine-5-prime-phosphorimidazolide is allowed to decompose in aqueous solution at room temperature and pH 7.2, depending on the buffer, 5-24 percent is converted to the 2-prime,5-prime-cyclic phosphate (V). Although the extent of cyclization is much greater than for adenosine-5-prime-phosphorimidazolide, cyclization is less efficient than hydrolysis and so would not substantially decrease the efficiency of condensation reactions in aqueous solution. The significance of this result for prebiotic chemistry is discussed.

Harada, Kazuo↗

NASA NTRS1990

Polymerization of the cyclic pyrophosphates of nucleosides and their analogues

When 2-prime-deoxythymidine 3-prime, 5-prime-cyclic diphosphate, or the cyclic pyrophosphates of the acyclic nucleoside analogs II and IV are heated to 65-85 C in the presence of imidazole, oligomers with lengths up to 20-30 are formed in excellent yield. This reaction provides a useful source of oligomers for use as templates in aqueous condensation reactions. In the absence of evidence to the contrary, it is assumed that the oligomers are atactic. The potential significance of this reaction in prebiotic chemistry is discussed.

Tohidi, Mahrokh↗

NASA NTRS1990

Polymerization of amino acids containing nucleotide bases

The nucleoamino acids 1-(3'-amino,3'-carboxypropyl)uracil (3) and 9-(3'-amino,3'-carboxypropyl)adenine (4) have been prepared as (L)-en-antiomers and as racemic mixtures. When 3 or 4 is suspended in water and treated with N,N'-carbon-yldiimidazole, peptides are formed in good yield. The products formed from the (L)-enantiomers are hydrolyzed to the monomeric amino acids by pronase. Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (L)-enantiomer of 3 did not act as templates to facilitate the oligomerization of 4.

Ben Cheikh, Azzouz↗

NASA NTRS1990

Template-directed oligomerization of 5-prime-deoxy 5-nucleosideacetic acid derivatives

5-prime-deoxy 5-nucleosideacetic acids H-II are isostructural analogs of nucleotides with a carboxylate group in the place of the 5-prime-phosphate group. In this work, their oligomerization in aqueous solution was studied using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogs IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.

Harada, Kazuo↗

NASA NTRS1990

Synthesis of 5'-deoxy-5'-nucleosideacetic acid derivatives

Several new 5'-deoxy-5'-nucleosideacetic acid derivatives have been synthesized by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5'-aldehydes. The oligomerization of adenine derivatives IIa, IIIa, IV, V and guanine derivatives IIc and IIIc in aqueous solution was studied using a water-soluble carbodiimide as a condensing agent. It is found that the saturated acid (IV) tends to cyclize to the lactone, while IIa and unsaturated acids (IIIa and V) oligomerized efficiently, especially in the presence of poly (U) as a template.

Harada, Kazuo↗

NASA NTRS1989

Some acyclic analogues of nucleotides and their template-directed reactions

Bismonophosphoimidazolides of acyclic analogues of guanosine IV and adenosine V were synthesized. They undergo oligomerization in the presence of complementary polynucleotide templates. Details of their synthesis and their subsequent template- and nontemplate-directed reactions are described, and their possible relevance to the origin of life is discussed.

Tohidi, Mahrokh↗

NASA NTRS1989

The origin of polynucleotide-directed protein synthesis

If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that at first the simple attachment of amino acids to the 2'(3') termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

Orgel, Leslie E.↗

NASA NTRS1988

Template-directed oligomerization of 3-isoadenosine 5'-phosphate

Template-directed oligomerization of an activated derivative of 3-isoadenosine 5'-phosphate (piA) on polyuridylic acid was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3'-5'-linked oligomers while the reaction of ImpA gives only 2'-5'-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.

Hill, Aubrey R., Jr.↗

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.↗