Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

Park, Jihyun; Lee, Seonghyun; Won, Nabin; Shin, Eunji; Kim, Soohyun; Chun, Min-Young; Gu, Jungyeun; Jung, Gun‐Young; Lim, Kwang‐il; Jo, Kyubong

doi:10.1039/c8an01426d

Cited by 17 publications

(13 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2C). This pattern is similar to previous results of AT-specific polypyrrole compound 6 and FP-DBPs of H-NS and HMG 11 . This pattern is even retained in concatenated λ-DNA molecules, which allows us to readily identify the number of monomers and their orientation (Fig.…”

Section: Resultssupporting

confidence: 92%

“…They have been applied for many genome projects to aid genome assembly with sequencing 7,8 , as well as to reveal large scale structural variations of the genome 9,10 . Single-molecule DNA maps have also been utilized for the rapid identification of DNA molecules without sequencing 11,12 . Alternatively, elongated DNA molecules provide a platform to visualize biochemical information, such as chemical modifications 13,14 and DNA damage at the single-molecule level 15–18 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer

Shin

Kim

Lee

et al. 2019

Sci Rep

Self Cite

View full text Add to dashboard Cite

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.

show abstract

Section: Resultssupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer

Shin

Kim

Lee

et al. 2019

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…In contrast, YOYO-1 stained DNA ( 5 in Figure 1) stretched up to 21.8 ± 0.72 µm ( X / L = 134% ± 4.4%), which is significantly larger than other stretches. However, it is notable that this stretch would be 88% ± 3% compared with Murade’s contour length (24.67 µm) for YOYO-1 stained DNA [28]. For comparison, we added DNA stretches stained by FP-DBPs (DNA binding peptides) from 6 to 10 in Figure 1 [21,27].…”

Section: Resultsmentioning

confidence: 99%

“…TAMRA-polypyrrole was synthesized in the lab as previously described [28,32]. Briefly, a computer-assisted Fmoc solid–phase synthesis of polyamides was performed from Fmoc-Py-oxime resin and cleavage from the resin was performed with 1 mL of 3-(dimethylamino) propylamine (Dp) or 3,3’-diamino-N-methyldipropylamine treatment for 3 h at 55 °C.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations

Lee

Kim

et al. 2018

Polymers

Self Cite

View full text Add to dashboard Cite

Large DNA molecules have been utilized as a model system to investigate polymer physics. However, DNA visualization via intercalating dyes has generated equivocal results due to dye-induced structural deformation, particularly unwanted unwinding of the double helix. Thus, the contour length increases and the persistence length changes so unpredictably that there has been a controversy. In this paper, we used TAMRA-polypyrrole to stain single DNA molecules. Since this staining did not change the contour length of B-form DNA, we utilized TAMRA-polypyrrole stained DNA as a tool to measure the persistence length by changing the ionic strength. Then, we investigated DNA stretching in nanochannels by varying the ionic strength from 0.06 mM to 47 mM to evaluate several polymer physics theories proposed by Odijk, de Gennes and recent papers to deal with these regimes.

show abstract

Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal‐Free Electro‐Stain Composed of DNA‐Binding Proteins and Synthetic Polymers

Noh,

Kang,

Heo

et al. 2024

Advanced Science

Self Cite

View full text Add to dashboard Cite

This paper presents the first scanning electron microscopy (SEM)‐based DNA imaging in biological samples. This novel approach incorporates a metal‐free electro‐stain reagent, formulated by combining DNA‐binding proteins and synthetic polymers to enhance the visibility of 2‐nm‐thick DNA under SEM. Notably, DNA molecules stain with proteins and polymers appear as dark lines under SEM. The resulting DNA images exhibit a thickness of 15.0±4.0 nm. As SEM is the primary platform, it integrates seamlessly with various chemically functionalized large surfaces with the aid of microfluidic devices. The approach allows high‐resolution imaging of various DNA structures including linear, circular, single‐stranded DNA and RNA, originating from nuclear and mitochondrial genomes. Furthermore, quantum dots are successfully visualized as bright labels that are sequence‐specifically incorporated into DNA molecules, which highlights the potential for SEM‐based optical DNA mapping. In conclusion, DNA imaging using SEM with the novel electro‐stain offers electron microscopic resolution with the ease of optical microscopy.

show abstract

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins

Abstract: Two-color DNA physical map for efficient identification of single DNA molecules.

Cited by 17 publications

References 19 publications

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations

Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal‐Free Electro‐Stain Composed of DNA‐Binding Proteins and Synthetic Polymers

Contact Info

Product

Resources

About