2P085
Structural Dynamics of Hairpin RNA Measured by 2D Fluorescence Lifetime
Correlation Spectroscopy(Molecular Spectroscopy Laboratory, RIKEN)○Chao-Han Cheng, Kunihiko Ishii, Tahei Tahara
Introduction
RNA folding kinetics has attracted much interest because it provides information about the complex
energy landscape on which multiple misfolding structures appear in competition with the most stablenative structure. However, the study of structural dynamics of RNA is still difficult, especially for thesecondary structure formation. In this work, we investigated a FRET-labeled hairpin RNA and compared
it with the DNA analogue to understand the formation dynamics of a hairpin structure of RNA.
Furthermore, we applied the 2D fluorescence lifetime correlation spectroscopy (2D FLCS)1-3 to clarify theorigins of this difference and tried to observe specific lifetime components and their interconversion.
Experiment
The hairpin RNA that we used is a single-stranded RNA labeled with 6-FAM and TAMRA (6-FAM5’-UUUAACC(U)18GGUU-3’-TAMRA). The DNA analogue has the same labeled dye molecules andbases as RNA but uracil is replaced by thymine. They were prepared in buffer solutions (10 mM Tris-HCl,
1 mM EDTA, BSA 0.01 %, pH 8.0) with various concentration of NaCl. 2D FLCS was measured by ahome-built system.1-3 Each photon was collected with absolute arrival time determined from the start ofexperiment (T) and the relative arrival time determined from the excitation laser pulse (t). The 2D
emission-delay correlation map M(∆T; t’, t”) was generated at a characteristic time interval ∆T between
two photons emitted at t’ and t”. The maximum entropy method (MEM) was utilized to convert 2D
emission-delay correlation maps to 2D lifetime correlation maps so as to obtain the correlation of isolatedspecies.

+

Normalized Intensity (a.u.)

Emission Intensity (a.u.)

1.2At 0.5 M [Na ]
1.0
0.8

DNA

0.6RNA

0.4
0.2

1.0+

hpDNA with various [Na ]
0.2 M
0.4 M

0.8+

hpRNA with various [Na ]
0.2 M
0.4 M

0.6
0.4
0.2
0.0

1020304050607080oTemperature ( C)

Fig. 1. Melting profiles of RNA and DNA. The emission
intensity at 520 nm is plotted against temperature.

024Time (ns)6810Fig. 2. Fluorescence lifetimes of RNA and DNA atvarious [Na+].

Results and Discussion
Fig. 1 shows the melting profiles described by the(a)Amplitude

emission intensity at 520 nm for RNA and DNA at 0.5
M [Na+] plotted against temperature, reflecting theS1
S2
S3

quenched fluorescence of donor due to FRET. The
curves present the difference melting temperature of510even with same [Na ], suggesting that there is at" (ns)1different response to Na between them.

S3) were observed. They correspond to the diagonal
peaks in 2D lifetime correlation maps at ∆T=20-30 µs
shown in Fig. 3(b). Because S3 was also observed indonor-labeled RNA, it is assigned to acceptor-missing
RNA. S1 shows short-lifetime components and is

6 7 8 923456 7 8 911086(b)
S3

864+

with 0.4 M [Na ]. Three isolated species (S1, S2, and52

difference also emerged in lifetime measurements+44

substantial difference in their structural change. TheFig. 3(a) shows the lifetime distribution of RNA

3Lifetime (ns)+shorter than DNA at same [Na+], indicative of the20.1

RNA and DNA. For RNA, it is 15 oC higher than DNA

shown in Fig. 2, where the lifetime of RNA is much

6 7 8 9S220.1

86S16 80.1

246 8

1t' (ns)

246 810Fig. 3. 2D FLCS measurement of RNA with 0.4 M[Na+]. The fluorescence lifetime distributions (a)and 2D lifetime correlation map (b) were evaluatedat ∆T=20-30 µs. Dashed squares corresponded tothe isolated species S1, S2, and S3.

assigned to the folded (F) form; moreover, the fast reaction among its components is observed, whichsuggests the formation and dissociation of the stem of RNA. S2 is assigned to the unfolded (U) form. Thisis because the lifetimes of components are strongly affected by Na+, implying a flexible structure such asa random coil. In the range of measurable delay time, the off-diagonal peaks between S1 and S2 were notobserved, indicating that the transition time between U and F forms of RNA is longer than ms, possibly
due to the rigid backbone which restricts the structural change.4 DNA, by contrast, shows a shortertransition time around 100 µs between U and F forms.3
In summary, the investigation of hairpin RNA shows the response to Na+ for the dynamics of RNA isdifferent from DNA. Furthermore, the results of 2D FLCS show that the transition time between F and U
forms for RNA is much longer than DNA, contributed from the difference in geometry.
Reference
1. K. Ishii and T. Tahara J. Phys. Chem. B 2010, 114, 12383.
2. K. Ishii and T. Tahara Chem. Phys. Lett 2012, 519-20, 130.
3. K. Ishii and T. Tahara Annual Meeting of Japan Society for Molecular Science 2011; K. Ishii and T.
Tahara, submitted for publication.
4. H. Chen, S. Meisburger, S. Pabit, J. Sutton, W. Webb, and L. Pollack PNAS 2012, 109, 799.