Disrupting ultrashort nucleic acid duplex with mechanical force

Speaker: Kevin Whitley

Department: University of Illionois at Urbana -Champain

Subject: Disrupting ultrashort nucleic acid duplex with mechanical force

Location: Bionanoscience Department, TU Delft

Date: 30-06-2017

Author: Mirte Golverdingen

Kevin Whitley did research to the hybridization kinetics of DNA under force. He tried to understand the transition state of nucleic acid hybridization. Hybridization is used for gene manipulation and targeting for DNA nanomachines, tweezers and bipadal walkers. Short strands (about 10 bp) show a ‘all of nothing’ behavior when hybridizing. Fist one or two base pairs bind and then all base pairs bind without stable intermediates. This describes a two-state reaction in a schematic energy landscape. The kinetics of any of these states is determined by the transition state between them. Whitley searched for the transition state between the bound and the unbound DNA.

To research the hybridization kinetics of the DNA the end-to-end extension of the DNA was researched. This is the length of the top of the energy to the bottom of the energy where the DNA is hybridized. Bell’s equation describes the energy barrier between the end-to-end extension. So, by measuring the rates of the Bell’s function you can calculate the end-to-end distance.

Whitley measured the hybridization rates by using optical beads that are bound to the DNA by double stranded handles and with a 9 nucleotides long middle strand. This short part is the binding site for short DNA. The distance between the two beads was approximately one. By using a flow chamber Whitley was able to look at oligo that were free in solution. He was able to measure the koff and kon rate under a constant amount of force. In this way, he could measure the moment a ss DNA oligomer binds to the middle strand by measuring the position of the bead. The ds DNA is stiffer than ss DNA, and therefore the position of the bead changes. By using this method, Whitley could measure the difference between ss and ds DNA up until a force of 12 pN.

By using Fleezers, which is a combination between fluorescence and optical tweezers, Whitley was able to label the oligomer with a fluorophore (see figure 1). In this way, also forces lower than 12 pN can be measured. Again, they calculated the binding and unbinding rates of the DNA. While the unbinding rate of the DNA goes up linearly when the force increases, the binding rate also goes up, however, not linearly. When the oligomer becomes longer, the unbinding rate goes down. For the binding rate no dependence on the length of the oligomer can be found.

Honours12

Figure 1: Measurement of single-oligonucleotide hybridization kinetics under force. (A) Schematic of the hybridization assay (not to scale). An engineered DNA molecule (red) containing a short, central ssDNA region flanked by long double-stranded DNA (dsDNA) handles is held under constant force by polystyrene beads (grey spheres) held in optical traps (orange cones). A fluorescence excitation laser (green cone) is focused on the central ssDNA region. Short oligonucleotides (blue) labeled with a Cy3 fluorophore at the 3΄ end (green disk) bind and unbind to the complementary ssDNA sequence in the center of the tethered DNA. The binding and unbinding is observed by the fluorescence emitted from the attached fluorophores.

Adapted from: Kevin D. Whitley, Matthew J. Comstock, Yann R. Chemla; Elasticity of the transition state for oligonucleotide hybridization. Nucleic Acids Res 2017; 45 (2): 547-555. doi: 10.1093/nar/gkw1173

When looking at Bell’s equation, the binding rate should be an exponential force-dependence, however, non-exponential behavior is reproducible across other conditions. The end-to-end distance is therefore not constant, and the Bells’ equation changes so an integral is used instead.  By calculating the unbound and bound extension by using the worm like chain model for double stranded DNA they were able to calculate the end-to-end extension. In this way, they also obtained the persistence length and contour length of the end-to-end extensions. He repeated these calculation for all different lengths of oligomers. The transitions state persistence length is comparable to ssDNA, however it is a bit stiffer.

The transition state is probably no random process, as simple polymer estimations predict a low probability of spontaneous alignments. The single strand that is a bit stiffer, as measured in the transition state, this could correspond to prearranging of the strands, prior to nucleation. Some enzymes can pre-organize mRNA so the transition state energy barrier becomes lower. Moreover, fast target finding is possible when there are preorganized bases by enzymes. In this way, they can speed hybridization up by pre-paying the entropy penalty.

Whitley told a clear story on obtaining the end-to-end distance of the DNA hybridization translation state. The combination of Optical Tweezers and fluorescence was very cool. Moreover, using the mathematics in combination with the biology inspired me as Nanobiology student.

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Optical Tweezers: gene regulation, studied one molecule at a time

Speaker:  Steven M. Block
Institute Speaker: Applied Physics and Biological Sciences, Stanford University
Organising Department: Kavli Institute
Subject:  Optical Tweezers: gene regulation, studied one molecule at a time    
Location: Faculty of Industrial Design, TU Delft
Date: 01-12-16

Author: Nemo Andrea

Professor Block is a well-known figure in the bionanoscience department. He is considered to be one of the founders of the single molecule biophysics field. He currently holds the Ascherman Chair in the Departments of Applied Physics and Biology at Stanford University. One of his former students is Elio Abbondanzieri, who is currently one of the members of the bionanoscience department at the TU Delft. He gave an intriguing seminar on Optical tweezers and their applications and discussed riboswitches.

In the introductory section, Professor Block discussed the importance of the single molecule biophysics field, and how bulk analysis can have it shortcomings. This was followed by a short section on one of the workhorses of this field: the optical trap. Having been introduced to the working concept and applications of these optical traps in our course, it was interesting to see someone who pioneered the use of optical tweezers.

The more contemporary discussion started with the versatility of RNA polymerase (RNAP), which coincidentally was also the focus of the preceding week’s BN seminar. This RNAP molecule, as he explained is a very well document and very versatile protein complex which is regulated in part by RNA hairpins. To demonstrate both the applications of optical tweezers and the properties of DNA he explained how they could determine how many DNA molecules were tethered in their optical trap by measuring the force-distance curves and comparing it to the mechanical properties of DNA. This is not a new experiment by any stretch of the imagination, but it is key in illustrating the use of the optical trap.

The second part of his seminar revolved around the research done in part by Elio, namely researching the manner in which RNAP moves along the DNA, in particular focusing on testing the validity of the inchworm model. He explained the particular circumstances that made that experiment particularly challenging. To hear about the complexity of environmental noise and finding new ways to minimise noise in experiments was refreshing and educational, as we often solely see elegant and low noise data but never hear about what problem solving steps were taken in the actual experiment that ultimately lead to said results.

The Final and main focus of his seminar revolved around RNA hairpins and riboswitches. He explained how various theoretical formulas can be used to determine many properties of various RNA hairpins. They studied riboswitches, with a particular focus on a riboswitch that as adenine as its substrate. The idea behind this particular type of riboswitch is that when the substrate binds to the RNA molecule, it will affect the transcription of the gene itself. This way we have a way in which RNA molecules can become part of the complex gene regulation networks. This concepts also ties in nicely with the RNA world hypothesis.

While this seems feasible in theory, it is not enough to just describe a model qualitatively in order for it to be accepted. It must be mentioned that this is still an area of active research and only three riboswitches have currently been studied in any detail. Steven Block and his lab and others set out to characterise the mechanical properties and kinetic behaviour of the adenine riboswitch, with the goal of being able to describe it more quantitatively.

They studied force extension curves of the specific riboswitch (which ultimately is just RNA being transcribed) by using optical tweezers. They used a framework (Dudko 2006) [3] to find the free energy of activation and characterise various other aspects of the conformations of this RNA molecule as it would be transcribed. This ultimately lead to the creation of a 5 state system as described in a paper by Greenleaf et al. (2008). This group created an energy landscape of this system which elegantly describes the behaviour of this system and the effect of adenine on the energy levels.

Left: the adenine riboswitch as depicted in [1], right: the TPP riboswitch as seen in [2]

They also ran experiments on a different riboswitch, one that is found in eukaryotes and functions with TPP as a substrate. Structural analysis of this molecule had led to the proposal of a model wherein two hairpins would (blue and red in the image above) would come together in order for the riboswitch to be switched on. In order to verify this hypothesis, they combined the optical trap with FRET. They labeled each arm and observed the intensity and wavelengths of light emitted. This allowed them to make a model which suggests that there are various conformations in which this riboswitch can reside. More can be read about this in reference [2].

It was intriguing to see the results and applications of optical tweezers, a topic we have covered in physics courses and are heralded as one of the prime example of the nanobiology field and the new technologies that are created when the worlds of molecular biology and physics meet. Additionally, I had never explored riboswitches in any great detail, making this topic a completely new area for me. While they may only be a uncommon way to regulate gene expression, the mechanisms through which they regulate the genome are very unique and certainly require further study. Even with the things above, the thing I enjoyed most was seeing how theory led to experiments which then led to new theories and models.

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692365/
[2]https://elifesciences.org/content/4/e12362
[3]http://journals.aps.org/prl/abstract/10.1103/PhysRevLett.96.108101