Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays

Functional DNA;Fluorescence resonance energy transfer (FRET);Protein microarray;Deoxyribonucleic acid (DNA)

He, Ying

关键词: Functional DNA;    Fluorescence resonance energy transfer (FRET);    Protein microarray;    Deoxyribonucleic acid (DNA);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/29571/He_Ying.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Fluorescence resonance energy transfer (FRET) is used to monitor the metal ion dependent conformational change of the UO22+ specific DNAzyme 39E. Inactive metal ions, Mg2+ and Zn2+ are found capable of inducing folding between two pairs of stems while the active ions UO22+ and Pb2+ are not able to bring about the folding in any pairs of the stems. By correlating the enzymatic cleavage results, the Mg2+ and Zn2+ induced folding exerts a counter-productive effecton the activities. This result contradicts with the normal productive role that Mg2+ plays in the activation of ribozyme functions. At reduced ionic strength, even UO22+ and Pb2+ can induce folding due to the nonspecific electrostatic interaction from their divalent metal ion nature. And the progression of Mg2+ and Na+ concentration leads to an inactive-active-inactive transition inthe DNAzyme function. Single molecule FRET is also used to obtain kinetics for low UO22+concentration at reduced imaging buffer strength. Together with the biochemical characterizationfor the catalytic cores and optimal working conditions, a thorough understanding of thisDNAzyme 39E is obtained, which might be helpful in the design of sensitive and selectiveDNAzymes.FRET is also used to gain step-wise kinetic information of adenosine aptamer structure-switching sensor. The kinetic information for individual steps (folding and releasing) obtained bymonitoring FRET process between fluorophores labeled at several positions of the aptamerstructure switching sensor provides direct evidence for sequential occurrences, as predicted by the structure switching principle. The information obtained here will facilitate sensors designed basedon the structure-switching principle.Electrohydrodynamic jet (E-jet) printing is applied to the protein microarray field. With the development of multi-nozzle printing system, both single-protein array and multiple-proteinarrays are successfully demonstrated. Several proteins, such as streptavidin, Green fluorescence protein (GFP), mCherry (a red fluorescence protein), et.al, have been printed, proving E-jet printing is generally applicable to many types of proteins. The printed streptavidin maintains itsbinding character with biotin, showing the printed streptavidin is still structurally intact andfunctionally active. What’s more, this printing technology has also been employed to immunology application. Immunoglobulin (Ig) G from several animal species are printed by E-jet printing, and their binding specificities to corresponding secondary antibody, anti IgG, are maintained. To prove this technique suitable for more practical applications, an inch-sized array with well controlled feature details has been completely in a short period of time. Overall, this technique will be a promising candidate for future protein microarray fabrication method.