Capillary electrophoretic separation‐based approach to determine the labeling kinetics of oligodeoxynucleotides

Kanavarioti, Anastassia; Greenman, Kevin L.; Hamalainen, Mark; Jain, Aakriti; Johns, Adam M.; Melville, Chris R.; Kemmish, Kent; Andregg, William

doi:10.1002/elps.201200214

Cited by 13 publications

(85 citation statements)

References 25 publications

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“…These attributes are (i) room temperature reactivity at mM concentration, (ii) no loss of label upon standing, (iii) superior selectivity for T over C by a factor of 27, (iv) undetectable degradation of the DNA strand under the labeling conditions, and (v) undetectable reactivity towards the purines (false positives). Most importantly OsBp reactivity towards T or C was found to be independent of composition, sequence, length, and secondary structure of the DNA strand, as shown in [30–32] and also the results herein. For example, oligothymidylates, such as T8, T15 and T20, exhibit fully labeled product formation that is identical to the rate of labeling of an oligoadenylate with one T or, even to osmylation of the monomer, the deoxythymidylic acid triphosphate, 5’dTTP [30].…”

Section: Introductionsupporting

confidence: 86%

“…Most importantly OsBp reactivity towards T or C was found to be independent of composition, sequence, length, and secondary structure of the DNA strand, as shown in [30–32] and also the results herein. For example, oligothymidylates, such as T8, T15 and T20, exhibit fully labeled product formation that is identical to the rate of labeling of an oligoadenylate with one T or, even to osmylation of the monomer, the deoxythymidylic acid triphosphate, 5’dTTP [30]. The across-the-board equal reactivity leads to predictable osmylation of any DNA without prior sequence knowledge.…”

Section: Introductionsupporting

confidence: 86%

“…Earlier studies identified a chromophore, resulting from the formation of the pyrimidine/OsBp bond, which absorbs in the range of 300 to 320 nm where DNA has practically no absorbance [30–32]. These studies also selected two wavelengths and their absorbance ratio ( R = A (λ = 312 nm)/ A (λ = 272 nm), i.e., the absorbance at 312 nm divided by the absorbance at 272 nm) as optimal for monitoring the osmylation of either T or T + C. Osmylation can be monitored at 312 nm directly, but determining R cancels sampling errors.…”

Section: Resultsmentioning

confidence: 99%

“…Conjugation of OsBp to a pyrimidine leads to a new chromophore that absorbs in the range of 300 to 320 nm, a range where DNA does not absorb. This last feature enabled the development of a quality control UV–vis assay to confirm the extent of labeling [30–32]. …”

Section: Introductionmentioning

confidence: 99%

“…It is only the final product that carries the strong absorbance in the range of 300 to 320 nm, and hence discriminates itself from the other components by UV–vis. Even though OsBp is a minor component [30], for simplicity, the OsBp is referred to the 1:1 labeling mixture. Hence, the OsBp concentration (listed under reaction conditions) refers to the initial concentration of each of the two components.…”

Section: Introductionmentioning

confidence: 99%

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False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2’-bipyridine

Kanavarioti¹

2016

Beilstein J. Nanotechnol.

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SummaryOsmium tetroxide 2,2’-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product, osmylated DNA, was proposed as a surrogate for nanopore-based DNA sequencing due to OsBp’s “perfect” label attributes. Osmylated deoxyoligos translocate unassisted and measurably slow via sub-2 nm SiN solid-state nanopores, as well as via the alpha-hemolysin (α-HL) pore. Both nanopores discriminate clearly between osmylated and intact nucleobase; α-HL was also shown to discriminate between osmylated T and osmylated C. Experiments presented here confirm that the kinetics of osmylation are comparable for short oligos and long ssDNA and show that pyrimidine osmylation is practically complete in two hours at room temperature with less than 15 mM OsBp. Under the proposed labeling conditions: deoxyoligo backbone degradation measures less than 1/1,000,000; false positives such as osmylated deoxyadenine (A) and osmylated deoxyguanine (G) measure less than 1/100,000; false negatives, i.e., unosmylated C measure less than 1/10,000; and unosmylated T must measure substantially lower than 1/10,000 due to the 27-fold higher reactivity of T compared to C. However, osmylated C undergoes degradation that amounts to about 1–2% for the duration of the labeling protocol. This degradation may be further characterized, possibly suppressed, and the properties of the degradation products via nanopore translocation can be evaluated to assure base calling quality in a DNA sequencing effort.

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Section: Introductionsupporting

confidence: 86%

Section: Introductionsupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2’-bipyridine

Kanavarioti¹

2016

Beilstein J. Nanotechnol.

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Diastereoselectivity of 5-Methyluridine Osmylation Is Inverted inside an RNA Chain

Tserovski

Helm

2016

Bioconjugate Chem.

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In this study, we investigated the reaction of the osmium tetroxide-bipyridine complex with pyrimidines in RNA. This reagent, which reacts with the diastereotopic 5-6 double bond, thus leading to the formation of two diastereomers, was used in the past to label thymidine and 5-methylcytosine in DNA. In light of the growing interest in post-transcriptional RNA modifications, we addressed the question of whether this reagent could be used for labeling of the naturally occurring RNA modifications 5-methylcytosine and 5-methyluridine. On nucleoside level, 5-methylcytosine and 5-methyluridine revealed a 5- and 12-fold preference, respectively, over their nonmethylated equivalents. Performing the reaction on an RNA level, we could show that the steric environment of a pentanucleotide has a major detrimental impact on the reaction rate of osmylation. Interestingly, this drop in reactivity was due to a dramatic change in diastereoselectivity, which in turn resulted from impediment of the preferred attack via the si side. Thus, while on the nucleoside level, the absolute configuration of the major product of osmylation of 5-methyluridine was (5R,6S)-5-methyluridine glycol-dioxoosmium-bipyridine, reaction with an RNA pentanucleotide afforded the corresponding (5S,6R)-diastereomer as the major product. The change in diastereoselectivity lead to an almost complete loss of selectivity toward 5-methylcytosine in a pentanucleotide context, while 5-methyluridine remained about 8 times more reactive than the canonical pyrimidines. On the basis of these findings, we evaluate the usefulness of osmium tetroxide-bipyridine as a potential label for the 5-methyluridine modification in transcriptome-wide studies.

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Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Sultan¹,

Kanavarioti²

2019

Sci Rep

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Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.

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Capillary electrophoretic separation‐based approach to determine the labeling kinetics of oligodeoxynucleotides

Cited by 13 publications

References 25 publications

False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2’-bipyridine

False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2’-bipyridine

Diastereoselectivity of 5-Methyluridine Osmylation Is Inverted inside an RNA Chain

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

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