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Abstract Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas.
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Impacts of sequence and structure on pyrrolocytosine fluorescence in RNA
Abstract Fluorescence spectroscopy encompasses many useful methods for studying the structures and dynamics of biopolymers. Applications to nucleic acids require the use of extrinsic fluorophores such as fluorescent base analogs (FBAs), which mimic the native bases but have enhanced fluorescence quantum yields. In this work, we use multiple complementary methods to systematically investigate the sequence- and structure-dependence of the fluorescence of the FBA pyrrolocytosine (pC) within RNA. We demonstrate that pC is typically brightest in conformations in which it is base-stacked but not base-paired, properties that distinguish it from more widely used FBAs. This effect is strongly sequence-dependent, with adjacent adenosine and cytidine residues conferring the greatest contrast between stacked and unstacked structures. Structural heterogeneity was resolved in single-stranded RNA and fully complementary and mismatched double-stranded RNA using time-resolved fluorescence measurements and fluorescence-detected circular dichroism spectroscopy. Double-stranded contexts are distinguished from single-stranded contexts by the presence of inter-strand energy transfer from opposing bases, while base-paired pC is distinguished by its short excited state lifetime. This work will enhance the value of pC as a structural probe for biologically and medicinally significant RNAs by guiding the selection of labeling sites and interpretation of the resulting data.
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- Award ID(s):
- 2338251
- PAR ID:
- 10673598
- Publisher / Repository:
- Nucleic Acids Research
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 53
- Issue:
- 7
- ISSN:
- 0305-1048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1093/nar/gkaf262
