Nanomechanics of D- and I-substituted DNA Restricted; Files Only

Zheng, Zixing (Spring 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/ht24wk572?locale=en
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Abstract

Deoxyribonucleic acid (DNA) is the molecule that carries genetic information for almost all living organisms. DNA exist in double-helical structure but there are evidence showing its polymorphism, and its conformation, as well as the physicochemical properties, can be affected by multiple factors. In this study, we focused on the relationship between specific properties of the individual bases, such as the number of hydrogen bonds they can establish in a base pair, and the biochemical characteristics of the whole double-helical structure. By substituting selected DNA sequences with either 2,6-diaminopurine(D) for adenine(A) or inosine(I) for guanosine(G), we investigated the influence of changing the number of hydrogen bonds, either increasing it to three or decreasing it to two throughout a molecule, without sacrificing sequence specificity. We used the melting temperature, CD spectroscopy and AFM imaging to characterize these effects. We found that D-substituted DNA has higher, and I-substituted DNA lower, melting temperature, compared to wild-type(WT) DNA. We found that We found that the hydrogen bonding is not the only factor influencing the stability of the DNA structure. CD spectra confirmed that both D-substituted and I-substituted DNA undergo some conformational change, but that overall, they are in the B-form. Furthermore, D-substituted DNA shows a progressive shift from a less-WT towards a WT-like spectrum with the increase of GC content, while the change is not significant for I-substituted DNA. The AFM images were used to visualize the DNA molecule under no tension. The contour length was recorded by tracing the molecules and from this parameter the axial rises per base pair was calculated. Compared to wild type DNA, D-substitution decreased the axial rise per base pair and I-substituted DNA increased it, which means D-substituted DNA is more compact than I-substituted DNA. 

Table of Contents

1 Introduction.............................................................................................1

2 Materials and Methods................................................................................6

2.1 Melting Temperature Characterization...................................................8

2.1.1 Sample Preparation................................................................8

2.1.2 Melting Temperature measurement ............................................8

2.2 Circular Dichroism Spectroscopy.........................................................8

2.3 Atomic Force Microscopy..................................................................8

2.3.1 Sample Preparation...............................................................8

2.3.2 AFM assay ........................................................................9

3 Results...................................................................................................10

3.1 Melting Temperature.......................................................................10

3.2 CD spectroscopy............................................................................15

3.3 AFM images.................................................................................22

4 Discussion and Conclusion.........................................................................27

5 References..............................................................................................29

6 Supplementary Table................................................................................36 

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