Steven A. Benner

The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system

Synthetic biology based on a six-letter genetic alphabet that includes the two non-standard nucleobases isoguanine (isoG) and isocytosine (isoC), as well as the standard A, T, G and C, is known to suffer as a consequence of a minor tautomeric form of isoguanine that pairs with thymine, and therefore leads to infidelity during repeated cycles of the PCR. Reported here is a solution to this problem. The solution replaces thymidine triphosphate by 2-thiothymidine triphosphate (2-thioTTP). Because of the bulk and hydrogen bonding properties of the thione unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. To test whether this might allow PCR amplification of a six-letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis. The concentrations of isoCTP and 2-thioTTP were optimal to best support PCR amplification using thermostable polymerases of a six-letter alphabet that includes the isoC–isoG pair. The fidelity-per-round of amplification was found to be ∼98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only ∼93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.

Redesigning the Molecules of Life

Our efforts on the modelling and redesign of proteins have proceeded along two general routes. In one approach, the principal subject of this article, we have focussed on designing and building model peptides where we could neglect tertiary structure in at least the early phases of our work. For many peptides and proteins that bind in the amphiphi1ic environments of biological interfaces, complementary amphiphi1ic secondary structures are induced. We have developed design principles for the construction of model peptides which have illuminated the roles of secondary structures in the biological activity of apolipoproteins, peptide toxins and peptide hormones. In the other approach to protein engineering that we have pursued, we have undertaken to redesign known tertiary structures by sitedirected mutagenesis or by chemical modification. In the latter work we have introduced new covalently bound coenzyme analogs into proteins, giving rise to semisynthetic enzymes with novel catalytic activities. Recently, we have embarked on a program of replacing secondary structural units in folded proteins of known tertiary structure by relatively non-homologous segments constructed employing design principles similar to those used in our amphiphi1ic pept:i,de work. These studies represent a first step towards the design and construction of tertiary structure by the assembly of primary sequences. s. A. Benner (Ed.) Redesigning the Molecules of Life

Redesigning nucleic acids

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.

A Strategy for Origins of Life Research

Contents 1.u2002Introduction 1.1.u2002A workshop and this document 1.2.u2002Framing origins of life science 1.2.1.u2002What do we mean by the origins of life (OoL)? 1.2.2.u2002Defining life 1.2.3.u2002How should we characterize approaches to OoL science? 1.2.4.u2002One path to life or many? 2.u2002A Strategy for Origins of Life Research 2.1.u2002Outcomes—key questions and investigations 2.1.1.u2002Domain 1: Theory 2.1.2.u2002Domain 2: Practice 2.1.3.u2002Domain 3: Process 2.1.4.u2002Domain 4: Future studies 2.2.u2002EON Roadmap 2.3.u2002Relationship to NASA Astrobiology Roadmap and Strategy documents and the European AstRoMap u2002Appendix I u2002Appendix II u2002Supplementary Materials u2002References

Reconstructing the Evolution of Proteins

Data from biological chemistry, including protein structurer enzymatic mechanisms, and metabolic pathways, can be unified by understanding the distinction between selected and non-selected behaviors. We outline methods for constructing functional and historical models explaining these behaviors, and show how they can be applied to organizing biochemical data, tested experimentally, and used to engineer the behavior of proteins using recombinant DNA techniques.