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Surface Modification for Two-End DNA Immobilization and Study of Single Molecule Dye, Probe, and DNA Interaction

Gautam, Dinesh
http://orcid.org/0000-0001-6205-0415
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1699531733320262

Abstract Details

2023, Doctor of Philosophy (PhD), Ohio University, Chemistry and Biochemistry (Arts and Sciences).
In recent years, there took place a notable advancement observed in single-molecule fluorescence microscopy methods and its use in various biomolecular research. This technique allows for direct visualization of dynamics and its detailed complexities of various biological processes at the molecular level which is not possible in bulk measurement. Usually, in experiments related to single-molecule fluorescence measurements, the abundance of key molecules is intentionally minimized, which reduces the noise and improves the quality of imaging. However, such a strategy does not work when experiments involve weak interaction between biomolecules. In such a situation nonspecific interaction between molecules of interest and glass would lead to an unwanted fluorescence background signal, which compromises the imaging quality and reduces the measurement accuracy. In this work, glass surfaces have been functionalized in multiple steps. In the initial step, the glass coverslip surface is modified with (3-aminopropyl) triethoxysilane (APTES), and in the next step, the surface is functionalized using methoxy-terminated polyethylene glycol (mPEG) and biotin-terminated polyethylene glycol (bPEG) molecules. Each surface is characterized using dye-labeled protein molecules called neutravidin. for a variety of single-molecule fluorescence studies as PEG molecules are known to repel any nonspecific molecules binding on the functionalized surfaces. Then the surface is used for two-end immobilization of lambda DNA using biotin and neutravidin interaction. Once the surface is functionalized and characterized, lambda DNA is two ends immobilized on the surface using biotin and neutravidin interaction. Then we use that platform to study the intercalation and de-intercalation kinetics of various intercalating dyes such as single- intercalator (YO-PRO-1) and doubleintercalator (YOYO-1) at various experimental conditions of ionic strength and flow speed of the buffer. Briefly, two different annealed and non-annealed samples of lambda DNA and intercalating dyes YO-PRO-1 and YOYO-1 were prepared by mixing dye and DNA at a 1:5 dye-to-base pair ratio before each experiment. The annealed sample was prepared by incubating the mixture at 50°C for more than 3 hrs. and the non-annealed sample was prepared by incubating at room temperature for more than ½ hrs. And biotin functionalized lambda DNA molecules which are stained with YO-PRO-1 and YOYO-1 were two ends immobilized on a glass surface functionalized with mPEG and bPEG using neutravidin and biotin interaction. Then intercalated dye molecules were washed from each immobilized DNA molecule in the channel by flowing buffer solution and studied the effect of buffer ionic strength and flow speed on dye desorption from DNA molecules. To further evaluate the surface functionalization and two-end DNA immobilization, short single-stranded triplex-forming oligonucleotides (TFO) were designed and used to analyze the efficiency of surface passivation for fluorescence single molecules studies and also tested their ability to triplex formation. Briefly, using the hope-built MATLAB function, short TFO probes of 4-10 base pairs were designed. In the first step, the surface is functionalized using mPEG and mPEG. Then, additional treatment of the surface is done using various agents such as short MS(PEG)4-NHS, nonionic detergent Tween 20, bovine serum albumin (BSA), and short single-stranded DNA molecules to enhance the treatment of surface for single molecules fluorescence research. We compare the efficiency of each method in reducing the nonspecific binding of dye-labeled TFO probes by tallying the number of particles per frame using the total internal reflection fluorescence (TIRF) microscope. Additionally, the surface with improved passivation is utilized to immobilize DNA and to measure the efficiency of TFO probes to form triplex with double-stranded lambda DNA at specific sequences. For this, DNA is immobilized on the surface and recorded its image and TFO probes flowed through the same channel and recorded the image of the probe at the same location of the DNA and overlap these two channels and evaluate the binding of TFO probes on the specific sequences along the DNA by using fluorescence microscopy and fluorometer for bulk measurements.
Jixin Chen (Advisor)
131 p.
Chemistry; Physical Chemistry
Lambda DNA; Neutravidin; YOYO-1; YOPRO-1; PEG; Biotin; Intercalation; Fluorescence; TIRF; Microscopy; Laser; BSA; Protein; Dye; mbPAINT; Detector

Recommended Citations

Citations

  • Gautam, D. (2023). Surface Modification for Two-End DNA Immobilization and Study of Single Molecule Dye, Probe, and DNA Interaction [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1699531733320262

    APA Style (7th edition)

  • Gautam, Dinesh. Surface Modification for Two-End DNA Immobilization and Study of Single Molecule Dye, Probe, and DNA Interaction. 2023. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1699531733320262.

    MLA Style (8th edition)

  • Gautam, Dinesh. "Surface Modification for Two-End DNA Immobilization and Study of Single Molecule Dye, Probe, and DNA Interaction." Doctoral dissertation, Ohio University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1699531733320262

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Ohio University and OhioLINK.