Exponential growth by cross-catalytic cleavage of deoxyribozymogens

Abstract

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...

Self-replication and Evolution of DNA Crystals

“…Other scientists have proposed that life might have originated with a set of molecules whose cross-catalytic activity would produce an autocatalytic cycle [FKP86,Wäc88,Wäc90]. While mathematical analysis of models of these systems has suggested that they have the ability to evolve [Eig71, Kau93, SBEL00], experimental demonstrations have been restricted to either systems for which catalysis does not form a complete cycle [HW], or to systems that contain autocatalytic cycles that do not obviously appear capable of non-trivial evolution [vK86,LGMKSa96,LE03]. The most well-accepted hypothesis about life that preceded the current DNA/RNA/protein life is the RNA world hypothesis, which proposes that genetic information was once stored by RNA molecules which could catalyze their own replication.…”

Systems Chemistry on Ribozyme Self‐Construction: Evidence for Anabolic Autocatalysis in a Recombination Network

“…Therefore, the entire network could be viewed as an "individual", in which specific sequences are required for collaboration to produce an autocatalytic network. This system provides an example of predicted missing links [9] between the first self-replicating nucleic acid templates and a more complex RNA world, in line with Eigens demarcation. Future work will examine whether collaborating molecules can display a selective advantage through autocatalysis and if a mutated population derived from these sequences can evolve over time.…”

“…Implementations are found in two recent schemes involving hybridization networks. [7,8] Genetic autocatalysis is usually attributed to self-replication (the autocatalytic transfer of information from templates to copies), and examples based on nucleic acids, [9][10][11][12][13][14] peptides, [15,16] and organic molecules [17] as templates have been studied. This approach was greatly influenced by Eigens description of the self-organization of matter that requires autocatalysis to evolve from a prebiotic "chemical" phase to a self-organization of replicating "individuals" phase, which must have occurred contemporaneously.…”

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

“…Although in principle ribozymes and deoxyribozymes could participate in similar cascades as catalysts [10][11][12], no generalizable method for implementing such cascades has yet been established. On the other hand, DNA and RNA can catalyze chemical reactions not only by forming intricate tertiary structures, but also by simply forming Watson-Crick base pairs.…”