title: Continuously tunable nucleic acid hybridization probes.
abstract: In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Lucia R
authors.initials: LR
authors.lastname: Wu
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.|Systems, Synthetic, and Physical Biology, Rice University, Houston, Texas, USA.
authors.forename: Juexiao Sherry
authors.initials: JS
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: John Z
authors.initials: JZ
authors.lastname: Fang
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Emily R
authors.initials: ER
authors.lastname: Evans
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pinto
authors.affiliation: Nanostring Technologies, Seattle, Washington, USA.
authors.forename: Irena
authors.initials: I
authors.lastname: Pekker
authors.affiliation: Nanostring Technologies, Seattle, Washington, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Boykin
authors.affiliation: Nanostring Technologies, Seattle, Washington, USA.
authors.forename: Celine
authors.initials: C
authors.lastname: Ngouenet
authors.affiliation: Nanostring Technologies, Seattle, Washington, USA.
authors.forename: Philippa J
authors.initials: PJ
authors.lastname: Webster
authors.affiliation: Nanostring Technologies, Seattle, Washington, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Beechem
authors.affiliation: Department of Bioengineering, Rice University, Houston, Texas, USA.|Systems, Synthetic, and Physical Biology, Rice University, Houston, Texas, USA.
authors.forename: David Yu
authors.identifier: http://orcid.org/0000-0002-0213-7663
authors.initials: DY
authors.lastname: Zhang
mesh_terms: D060885:Multiplex Polymerase Chain Reaction; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015341:Nucleic Acid Probes / Q000737:chemistry* / Q000235:genetics; D009696:Nucleic Acids / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D015341:Nucleic Acid Probes; D009696:Nucleic Acids