Parabolic Growth of a Self‐Replicating Hexadeoxynucleotide Bearing a 3'‐5'‐Phosphoamidate Linkage

Kiedrowski, Günter von; Wlotzka, Britta; Helbing, Jörg; Matzen, Matthias; Jordan, Stephan

doi:10.1002/anie.199104231

“…The self-replicating systems that have been studied to date use template molecules composed of nucleic acids (6,7,10), peptides (11)(12)(13)(14), or small organic compounds (15)(16)(17). The nucleic acid-based systems are the most straightforward and rely on simple Watson-Crick pairing interactions between a short oligonucleotide template and two complementary oligonucleotide substrates (6,7,10).…”

mentioning

confidence: 99%

“…The nucleic acid-based systems are the most straightforward and rely on simple Watson-Crick pairing interactions between a short oligonucleotide template and two complementary oligonucleotide substrates (6,7,10). The substrates are bound at adjacent positions along the template and are joined through a reaction involving chemical groups at their opposed ends.…”

mentioning

confidence: 99%

A self-replicating ligase ribozyme

Paul

¹

,

Joyce²

2002

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 min ؊1 , whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 ؋ 10 ؊11 M⅐min ؊1 . Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication. In living systems, replicative processes transfer genetic information from template nucleic acid molecules to newly synthesized, complementary products. Several nonenzymatic templatedependent ligation systems have been devised to study the role of a template in binding and positioning complementary substrates for covalent bond formation (1-5). These have included simple self-replicating systems of the form A ϩ B 3 T, where A and B are substrates that bind to a complementary template, T, and become joined to form a product molecule that is identical to the template (6-9). The unique aspect of self-replicating systems is that the reaction product has the potential to direct additional reactions. The system is termed autocatalytic when the product is an efficient template, and each covalent bond that is formed generates additional template molecules that can direct further joining reactions. The realization of autocatalytic behavior in a self-replicating system implies that sustained exponential growth may be possible.The self-replicating systems that have been studied to date use template molecules composed of nucleic acids (6, 7, 10), peptides (11-14), or small organic compounds (15-17). The nucleic acid-based systems are the most straightforward and rely on simple Watson-Crick pairing interactions between a short oligonucleotide template and two complementary oligonucleotide substrates (6, 7, 10). The substrates are bound at adjacent positions along the template and are joined through a reaction involving chemical groups at their opposed ends. Peptide-based self-replicating systems are similar, except that the components are oligopeptides that have the capacity to form ␣-helices. The template is the hydrophob...

show abstract

“…Variations on this theme have led to proof that short oligonucleotide templates are capable of semiconservative (cross-catalytic) replication (3), and that substrates as short as single nucleotides can be used (4-6). However, because of the strong interactions that exist between complementary strands, the products formed in these ligation-based systems bind strongly to one another and the reaction kinetics of these systems are limited to parabolic, rather than exponential, growth (7,8).Various strategies have been implemented to avoid the problem of product inhibition. For example, Li and Nicolaou (9) used cyclic pH changes and the addition of excess complementary oligonucleotide to ensure the release of ligated products from a template.…”

mentioning

confidence: 99%

“…Variations on this theme have led to proof that short oligonucleotide templates are capable of semiconservative (cross-catalytic) replication (3), and that substrates as short as single nucleotides can be used (4)(5)(6). However, because of the strong interactions that exist between complementary strands, the products formed in these ligation-based systems bind strongly to one another and the reaction kinetics of these systems are limited to parabolic, rather than exponential, growth (7,8).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Exponential growth by cross-catalytic cleavage of deoxyribozymogens

Levy

¹

,

Ellington

²

2003

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have designed an autocatalytic cycle based on the highly efficient 10 -23 RNA-cleaving deoxyribozyme that is capable of exponential amplification of catalysis. In this system, complementary 10 -23 variants were inactivated by circularization, creating deoxyribozymogens. Upon linearization, the enzymes can act on their complements, creating a cascade in which linearized species accumulate exponentially. Seeding the system with a pool of linear catalysts resulted not only in amplification of function but in sequence selection and represents an in vitro selection experiment conducted in the absence of any protein enzymes. D emonstrating molecular self-amplification is essential for understanding origins and can potentially foment biotechnology applications, especially in diagnostics. In modern biology, molecular replication is dominated by cycles in which nucleic acids encode and are replicated by protein enzymes. However, the discovery and subsequent engineering of nucleic acid catalysts raises the possibility that nucleic acid-based, autocatalytic cycles might be designed.In this regard, von Kiedrowski (1) and Zielinski and Orgel (2) showed that oligonucleotide palindromes can serve as templates for the ligation of shorter oligonucleotide substrates, and thus for their own reproduction. Variations on this theme have led to proof that short oligonucleotide templates are capable of semiconservative (cross-catalytic) replication (3), and that substrates as short as single nucleotides can be used (4-6). However, because of the strong interactions that exist between complementary strands, the products formed in these ligation-based systems bind strongly to one another and the reaction kinetics of these systems are limited to parabolic, rather than exponential, growth (7,8).Various strategies have been implemented to avoid the problem of product inhibition. For example, Li and Nicolaou (9) used cyclic pH changes and the addition of excess complementary oligonucleotide to ensure the release of ligated products from a template. The von Kiedrowski lab (10) facilitated product release by template immobilization and denaturation. Cycles of immobilization and denaturation resulted in the ''SPREAD'' (surface-promoted replication and exponential amplification) of oligonucleotides beyond an initial, immobilized population. Those experiments have yielded one of the first convincing demonstrations of the possibility of primordial Darwinian sequence selection. Most recently, Paul and Joyce (11) designed an autocatalytic cycle based on the R3C RNA ligase ribozyme. The self-reproducing ligase used ''half-ribozymes'' as substrates and was capable of limited exponential growth. This replicator appears to have neatly avoided the problem of product inhibition by refolding and forming more stable intramolecular contacts within the newly ligated product. However, the replicator did not show extensive exponential growth, as substrate self-association resulted in the accumulation of stable complexes that could not bind to the ribozyme ca...

show abstract