Selection of self-priming molecular replicators.

Self-priming amplification of oligonucleotides is possible based on foldback of 3' ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.

Coupling two different nucleic acid circuits in an enzyme-free amplifier.

DNA circuits have proven to be useful amplifiers for diagnostic applications, in part because of their modularity and programmability. In order to determine whether different circuits could be modularly stacked, we used a catalytic hairpin assembly (CHA) circuit to initiate a hybridization chain reaction (HCR) circuit. In response to an input nucleic acid sequence, the CHA reaction accumulates immobilized duplexes and HCR elongates these duplexes. With fluorescein as a reporter each of these processes yielded 10-fold signal amplification in a convenient 96-well format. The modular circuit connections also allowed the output reporter to be readily modified to a G-quadruplex-DNAzyme that yielded a fluorescent signal.