To test whether the presence of RNA in DNA duplexes could alter the elasticity and structure of DNA, a group of researchers at Georgia Tech and Georgia State University, inspired by Francesca Storici, and including the labs of Elisa Riedo, Angelo Bongiorno and Markus Germann conducted a multidisciplinary study at the interface of physics, chemistry and molecular biology. The group employed atomic force microscopy (AFM)-based single molecule force-measurements of short rNMP(s)-containing oligonucleotides in combination with molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR).

Ribonucleotides (rNMPs), the units of RNA, are the most abundant non-canonical nucleotides found in genomic DNA. rNMPs, either not removed from Okazaki fragments during DNA replication or incorporated and scattered throughout the genome, pose a perturbation to the structure and a threat to the integrity of DNA. The instability of DNA is mainly due to the extra 2’-hydroxyl (OH) group of rNMPs which gives rise to local structural effects that may disturb various molecular interactions in cells. As a result of these structural perturbations by rNMPs, the elastic properties of DNA may also be affected.

DNA has unique mechanical properties that are crucial in many natural biochemical processes and play an important role in DNA-based nanotechnology applications. Despite demonstrations of their abundance and importance, no data exist in literature regarding elastic measurements and sequence-dependent structural distortions of DNA with isolated single rNMP intrusions. With the goal to bring insights on how rNMPs change elastic properties of DNA and its structure, the Georgia Tech team with Hsiang-Chih Chiu and Kyung Duk Koh, a postdoctoral fellow at the time in the lab of Elisa Riedo in the School of Physics, and a PhD candidate in the lab of Francesca Storici from the School of Biology, respectively, together with the graduate student Annie Lesiak from Angelo Bongiorno lab in the School of Physics and School of Chemistry and Biochemistry, and in collaboration with Markus Germann and his graduate student Marina Evich from the Department of Chemistry at Georgia State University, conducted an innovative, experimental and theoretical study utilizing two short DNA molecules containing isolated rNMP intrusions. Storici said: <<We examined and identified how the elasticity and structure of DNA are altered by the rNMP intrusions in the studied DNA sequences>>. AFM-based single molecule force spectroscopy demonstrated that rNMP intrusions in short DNA duplexes can decrease – by 32% – or slightly increase the stretch modulus of DNA depending on specific sequence contexts next to the rNMPs. In addition, MD simulations and NMR experiments indicated that rNMP inclusions locally change the torsional distortion of the sugar-phosphate backbone in DNA only when the rNMPs are in specific locations in the DNA sequence. Riedo concluded: <<Our work opens up the route to use AFM single molecules measurements to understand how defects and the base sequence can affect the elasticity of short DNA molecules>>.

The demonstrated ability of rNMPs to locally change DNA mechanical properties and structure may find applications in structural DNA nanotechnology and help understanding how such intrusions impact DNA biological functions. Overall, these findings open a new route for understanding how rNMPs may influence DNA structure, chemistry, and biology.

The study is just published as an article in the journal Nanoscale (accepted, 2014):

Chiu HC*, Koh KD*, Evich M, Lesiak AL, Germann MW, Bongiorno A, Riedo E, Storici F (2014)

RNA intrusions change DNA elastic properties and structure. Nanoscale, DOI: 10.1039/C4NR01794C; *equal contribution.

http://pubs.rsc.org/en/content/articlepdf/2014/nr/c4nr01794c

This project was supported by the Office of Basic Energy Sciences of the US Department of Energy (DE-FG02-06ER46293), the National Science Foundation (NSF)(CMMI-1100290 and DMR-0820382), the Samsung Advanced Institute of Technology and the NSF grant CHE-0946869, the Integrative Biosystems Institute grant IBSI-4, the Georgia Research Alliance grant R9028 and the NSF grant MCB-1021763.