The following single molecule experiment setup was used to investigate helicase
ID: 174598 • Letter: T
Question
The following single molecule experiment setup was used to investigate helicase activity: A oligomeric DNA duplex was synthesized with a biotin label attached to the 3' end of one strand for binding the duplex to a PEG-coated surface that has been fractionally functionalized with streptavidin. The 5' end of the same strand has the fluorescent acceptor dye Cy5. On the opposite strand, the Cy3 donor is attached, followed by a T_20 tail for helicase binding. The helicase single molecule FRET (smFRET) experiment is carried out several times and two different results are observed, as shown in figures A and B. (a) For each of the two cases, explain what is happening that gives rise to the corresponding plot. (E_FRET = FRET efficiency) (b) Give two explanations for the results shown in figure B. How could between these two possibilities? c) What would you have expected to see had this been an ensemble experiment?Explanation / Answer
FRET efficiency is the quantum yield of the energy transfer transition or the fraction of the energy transfer event occurring per one donor excitation event.
a)
In the first figure A, the FRET or fluorescence resonance energy transfer gradually decreased indicating that the quantum yield of the energy transfer or the FRET efficiency reduced gradually.
In the figure B, the FRET efficiency reduced, remained at a level for some time and gradually increased. This graph indicates that the helicase activity reduced (retaining double strand DNA) and then gradually increased enhancing the separation of double stranded DNA into single strands.
b)
The helicase activity according to the second graph indicates that the double stranded DNA breaks down into single strand when the enzyme activity increased and retains as double stranded when the enzyme activity is reduced.
The overall figure shows the decrease, neutrality and increase of FRET efficiency. But, when the graph is enlarged, the decrease and increase of FRET efficiency is observed repeatedly indicating that the helicase activity also reduced and increased repeatedly.
Experimentally, this is possible when the excitation of the donor by the fluorescence light wavelength occurs accordingly. The higher wavelength light will have less frequency and viceversa. FRET efficiency would decrease with the less donor excitation with low frequency radiation.
C)
The ensemble experiment would result in detecting the DNA sequence used in the experiment and the database source of that DNA molecule. The source DNA molecule sequence that is actually responding to the current FRET experiment can be identified.