Selection, development and structural characterization of 2' FY-RNA aptamers for pyoverdine

Pyoverdines are siderophores employed by a variety of pseudomonas species to capture essential iron by which their growth is supported in many environments in which they can be harmful or helpful to coinhabiting species or the organisms that they inhabit. Of the Pseudomonas fluorescens group, the pyoverdine of each pseudomonad species is constituted of a central chromophore and a unique peptide component that is produced by nonribosomal peptide synthetases. Pyoverdine Pf5 (PYO-Pf5) is the product of pseudomonas protegens, a species that supports plant growth, particularly in soils with limiting iron. To explore the dynamics of P. protegens activity in soil, we have selected 2' FY modified RNA aptamers that selectively recognize PYO-Pf5 with high affinities. Two 2'FY-RNA aptamers, with different base sequences, were selected that specifically bind pyoverdine-Pf5 (PYO-Pf5) over pseudobactin (PSB), a related pyoverdine, and two other siderophores, enterobactin and ornibactin. The 2'FY-RNA aptamers were shown to be G-quadruplexes by computational prediction, N-methyl mesoporphyrin IX (NMM) binding and CD spectroscopy. They were compared with their counterparts (same base sequence) in RNA and DNA for function and structure. Whereas the 2'FY-RNA and RNA forms bound PYO-Pf5 and had circular dichroism (CD) spectra typical of a G-quadruplex, the DNA forms neither bound PYO-Pf5 nor showed diagnostic NMM binding or CD spectra for G-quadruplexes. The impact of 2' moiety in aptamers on their structure and function have been explored and results revealed that the nature of the chemical group at the 2' position influences binding activity and structure.The aptamers will be applied to a nanoporous aluminum oxide biosensor to detect PYO-Pf5. This sensor provides a future opportunity to track the locations around plant roots of P. protegens and to monitor PYO-Pf5 production and movement through the soil. Overall design: SELEX was performed with PYO-Pf5 as target to select 2'FY-RNA aptamers. For the first round of selection libraries of 1015 2' FY-RNA were used. The biotinylated capture oligos, which are complementary to the short constant region in the library sequence, were attached to streptavidin coated magnetic beads. The 2' FY-RNA libraries were first hybridized with oligos complementary to the PCR primer regions and then immobilized on the beads by hybridization with the capture oligos. The length of the capture oligo varied with the SELEX round as did the hybridization temperature and the oligo: PYO-Pf5 ratio . The bound oligos were captured using a magnet and purified by ethanol precipitation in the presence of 2 µL linear polyacrylamide (LPA) . Purified 2'FY-RNA was reverse transcribed and amplified by low fidelity Taq polymerase. The ratio of 2'FY-RNA to PYO-Pf5 was reduced and the length of capture oligo increased in successive SELEX rounds to increase selection pressure . Nine rounds of selection were performed, and two sequential rounds of counter selection were done after rounds 3 and 6. The targets for counter selection were enterobactin and ornibactin. All targets used in selection and counter-selection were the Fe3+-bound forms. PCR-amplified libraries were prepared for NGS using Nextera Illumina sequencing primers and the appropriate bar coding. Next generation sequencing was performed by Novogene facility, Sacramento, California using HiSeq PE150 platform. The raw NGS data was demultiplexed and assigned to respective rounds by open-source software AptaSuite. Barcodes were used to assign DNA pools from different rounds of selection. A zero-nucleotide mismatch between the reference and matched barcodes, and two mismatches between the reference and matched primers were criteria for demultiplexing. Further analysis was done with the sequences that met this standard. Clustering was done with 10 iterations of locality sensitive hashing, sampling 55% of the indices in the randomized regions and allowing no more than 10 nucleotide mismatches between the seed sequence of a cluster to each remaining member. Clusters were extracted for further analysis and aptamer screening.