Abstract
SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be 2-N-(3-dimethylaminopropyl)-N-propylamino]-4-2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenyl-quinolinium and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5~μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5~μM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.
Item Type: | Journal article |
---|---|
EU Funded Grant Agreement Number: | 638536 |
EU Projects: | Horizon 2020 > ERC Grants > ERC Starting Grant > ERC Grant 638536: SM-IMPORT - Substrate import at work: single-molecule studies of ABC transporters |
Faculties: | Physics Biology > Department Biology I |
Subjects: | 500 Science > 570 Life sciences; biology |
URN: | urn:nbn:de:bvb:19-epub-77650-1 |
Language: | English |
Item ID: | 77650 |
Date Deposited: | 26. Oct 2021, 17:56 |
Last Modified: | 03. Jan 2022, 18:12 |