Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.

We have made the surprising discovery that the crystallinity of nanoparticles formed from solutions containing RNA depends upon the presence of sequence mixtures. That is, a single sequence selected from the original random RNA sequence library produced mostly amorphous hexagonal nanoparticles, while a combination of sequences that emerged from the selection yields crystalline material as determined by SAED. To our knowledge this is the first example in which two biomolecules (RNA, DNA, or peptides) selected in vitro work together to provide a unique chemical outcome. In addition, this article provides a rigorous examination of the chemistry of Pd nanoparticle formation using RNA and the organometallic precursor complex Pd2(DBA)3 (DBA is dibenzylideneacetone). These studies have identified the specific conditions required for the successful RNA-mediated synthesis of Pd nanoparticles from aqueous solutions (10% THF:90% H2O) containing Pd2(DBA)3, as well as conditions that led to anomalous results. A variety of techniques were employed to characterize materials formed under different solution conditions including SEM, AFM, TEM, selected area electron diffraction (SAED), and a chemical reactivity test. These analysis methods support the formation of Pd particles by RNA mediation when accounting for and controlling the important variables in the execution of the experiments. It is now clear that nanoparticles formed from RNA sequences isolated viain vitro selection can be dependent on many factors and it is understood that the specific sequence or sequence mixtures must be taken into account to fully understand RNA mediation of nanoparticle formation.

In vitro selection experiments involving RNA, phagemids, or whole cells can yield biomolecules that bind tightly to or mediate the formation of inorganic materials. Herein we show that RNA sequences that mediate the formation of metal oxide nanoparticles can be isolated from iterative cycles of RNA selection and amplification. In contrast to prior work, the in vitro selection described within was based upon a desired materials property. In order to be isolated from a starting random sequence pool, an RNA sequence was required to mediate the assembly of Co and/or Fe into a solid that responded to a magnetic field. Sequences isolated from this selection were able to mediate the formation of iron oxide nanoparticles containing small amounts of Co under atypical synthesis conditions of temperature and pH.

Biopolymers in the biosphere are well known to mediate the formation of a wide array of inorganic materials, such as bone, shells, lenses, and magnetic particles to name a few. Recently, in vitro experiments with biopolymers such as peptides, RNA, and DNA have shown that templating by these macromolecules can yield a variety of materials under mild reaction conditions. The primary sequence of the biopolymer can be viewed as a proteomic or genomic signature for the templating of an inorganic material from defined metal precursors and reaction conditions. Together with the rapid advances in inorganic particle synthesis by other combinatorial methods, these bioinspired in vitro materials experiments may provide additional insights into possible inorganic materials yet to be discovered and subsequently synthesized by conventional methods. Some of the concepts important to understanding the crystallization phenomena occurring during biopolymer mediation are discussed. A simple kinetic model is provided in the context of known biopolymer-mediated inorganic crystallizations.Keywords: biomineralization; crystallization; DNA; mediation; nanocrystal; nanoparticle; phage display; RNA; SELEX

We have made the surprising discovery that the crystallinity of nanoparticles formed from solutions containing RNA depends upon the presence of sequence mixtures. That is, a single sequence selected from the original random RNA sequence library produced mostly amorphous hexagonal nanoparticles, while a combination of sequences that emerged from the selection yields crystalline material as determined by SAED. To our knowledge this is the first example in which two biomolecules (RNA, DNA, or peptides) selected in vitro work together to provide a unique chemical outcome. In addition, this article provides a rigorous examination of the chemistry of Pd nanoparticle formation using RNA and the organometallic precursor complex Pd2(DBA)3 (DBA is dibenzylideneacetone). These studies have identified the specific conditions required for the successful RNA-mediated synthesis of Pd nanoparticles from aqueous solutions (10% THF:90% H2O) containing Pd2(DBA)3, as well as conditions that led to anomalous results. A variety of techniques were employed to characterize materials formed under different solution conditions including SEM, AFM, TEM, selected area electron diffraction (SAED), and a chemical reactivity test. These analysis methods support the formation of Pd particles by RNA mediation when accounting for and controlling the important variables in the execution of the experiments. It is now clear that nanoparticles formed from RNA sequences isolated viain vitro selection can be dependent on many factors and it is understood that the specific sequence or sequence mixtures must be taken into account to fully understand RNA mediation of nanoparticle formation.

In vitro selection experiments involving RNA, phagemids, or whole cells can yield biomolecules that bind tightly to or mediate the formation of inorganic materials. Herein we show that RNA sequences that mediate the formation of metal oxide nanoparticles can be isolated from iterative cycles of RNA selection and amplification. In contrast to prior work, the in vitro selection described within was based upon a desired materials property. In order to be isolated from a starting random sequence pool, an RNA sequence was required to mediate the assembly of Co and/or Fe into a solid that responded to a magnetic field. Sequences isolated from this selection were able to mediate the formation of iron oxide nanoparticles containing small amounts of Co under atypical synthesis conditions of temperature and pH.