title: Capillary electrophoretic separation-based approach to determine the labeling kinetics of oligodeoxynucleotides.
abstract: With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined CE protocol to assess the label stability, reactivity, and selectivity. We report our protocol using osmium tetroxide 2,2'-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence, the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in ssDNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM.
authors.affiliation: Halcyon Molecular, Inc., Redwood City, CA, USA. tessi.kanavarioti@gmail.com
authors.forename: Anastassia
authors.initials: A
authors.lastname: Kanavarioti
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Greenman
authors.forename: Mark
authors.initials: M
authors.lastname: Hamalainen
authors.forename: Aakriti
authors.initials: A
authors.lastname: Jain
authors.forename: Adam M
authors.initials: AM
authors.lastname: Johns
authors.forename: Chris R
authors.initials: CR
authors.lastname: Melville
authors.forename: Kent
authors.initials: K
authors.lastname: Kemmish
authors.forename: William
authors.initials: W
authors.lastname: Andregg
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D016014:Linear Models; D019906:Nuclear Magnetic Resonance, Biomolecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013936:Thymidine / Q000737:chemistry
chemical_list: D009838:Oligodeoxyribonucleotides; D009942:Organometallic Compounds; D011725:Pyridines; C092906:osmium tetroxide bispyridine; D013936:Thymidine