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2008, 112, 1336-1338 Published on Web 01/16/2008
Evidence for Coupling between Nitrile Groups Using DNA Templates: A Promising New Method for Monitoring Structures with Infrared Spectroscopy Amber T. Krummel and Martin T. Zanni* UniVersity of WisconsinsMadison, Department of Chemistry, 1101 UniVersity AVenue, Madison, Wisconsin 53703 ReceiVed: December 7, 2007
Infrared spectroscopy is a common method for monitoring biomolecular structures but suffers from spectral congestion. Non-natural vibrational probes provide a way to regain structural specificity because they provide a unique vibrational signature and can be incorporated into proteins or other biomolecules at specific locations. A popular probe is the nitrile group because its frequency is sensitive to the electrostatics of its environment. In this work, we show that pairs of nitrile groups can be used to directly probe distances and angles in dual labeled molecules. By labeling model DNA oligomers with pairs of nitrile tags, we demonstrate that the vibrational coupling between two nitrile groups is strong enough that Fourier transform infrared (FTIR) spectra can be used to probe relative nitrile distances >4.5 Å. Our approach is similar in spirit to monitoring structures with fluorescence resonance energy transfer (FRET) using a pair of fluorescent labels or a pair of spin labels in electron spin resonance spectroscopy. The small sizes of nitrile groups make especially valuable probes of sterically confined regions like the inner cores of large biomolecules where other spectroscopic probes do not fit.
Non-natural vibrational probes provide a way to gain structural specificity with infrared spectroscopy. One very useful probe is the nitrile group, because it has a unique absorptive signature and can be incorporated into proteins at specific locations. Nitrile groups have been utilized to study the electrostatic environments of proteins,1 ligand binding,2 the orientations of membrane peptides,3 the structures of amyloid fibers,4 the folding kinetics of peptides,5 and, most recently, the Stark effect in DNA.6 The frequency of the nitrile tag reports on its local environment but does not directly monitor protein conformation because the nitrile vibrational mode is very weakly coupled to the protein backbone and side chains. Thus, frequency shifts of single nitrile labels can be insensitive to many types of structural changes where little variation in environment occurs. In this paper, we take a complimentary approach by using pairs of nitrile labels. Using nitrile labeled DNA oligomers as a model system, we present evidence that the coupling between nitrile tags is substantial enough that pairs of nitrile groups can be vibrationally coupled at distances >4.5 Å. Our approach is similar to using a FRET pair or two electron spin labels to measure distances but has the advantage that the small size of nitrile groups allows them to be incorporated into much more sterically confined locations.7 With pairs of nitrile tags, it should be possible to directly probe structures in addition to the environments of very large biomolecules using IR spectroscopy. A schematic of our approach is shown in Figure 1. We synthesized two noncomplimentary DNA oligomers with nitrile groups. On the 3′ end of one oligomer, we attached a C14NdU base. At the 5′ end of the other oligomer, we attached a base with an isotope labeled nitrile, C15NdU. An equal mixture of * To whom correspondence should be addressed. E-mail: zanni@ chem.wisc.edu.
10.1021/jp711558a CCC: $40.75
Figure 1. Schematic of the experimental approach. Two noncomplimentary oligomers are nitrile labeled with either C14NdU or C15NdU. After annealing with a third complimentary oligomer, the dsDNA is either B-form or A-form depending on the solvent conditions, stacking the labels adjacent to each other in the geometry shown at the bottom.
these two oligomers acts as a control, since these two oligomers do not associate and thus the two nitriles will not be coupled (a second control is reported in the Supporting Information). Upon addition of a template oligomer that is complimentary to the two labeled strands, a double helix forms, bringing the two labeled bases adjacent to each other. Nitrile groups are readily incorporated into DNA with no effect on the secondary structure.6,8 Therefore, according to standard models for DNA secondary structure, in B-form DNA, these two labels will be separated by RB ) 4.5 Å and lie at an angle of θB ) 35.8°, while, in A-form, they will have RA ) 3.8 Å and θA ) 32.7°. Thus, we can adjust the relative distances, and hence coupling strengths, of the two nitrile labels by choosing the secondary structure of the DNA template. © 2008 American Chemical Society
Letters
Figure 2. FTIR spectra of nitrile labeled DNA. ssDNA control containing C14NdU or C15NdU exhibiting absorption bands with equal intensities (a). Nitrile labeled dsDNA in the B-form (b) and A-form (c).
The nitrile labels were incorporated into DNA using protocols similar to Sigurdsson et al.8 Our exact procedure is given in the Supporting Information along with the DNA sequences and melting temperatures. Purified samples were either reconstituted in H2O or a solvent mixture containing 80% trifluoroethanol (TFE)/20% H2O (v/v) to induce an A-form helicity, which was confirmed with electronic circular dichroism (UVCD).9 All infrared spectra were recorded on a Vertex 70 Fourier transform infrared (FTIR) spectrometer (Bruker Optics, Inc.). Samples were held between two CaF2 plates with a 25 or 56 µm spacer. FTIR spectra of samples in H2O were recorded at ambient temperature (∼22 °C) and at 10 °C in TFE/H2O so that >95% of the oligomers are double stranded. The DNA samples ranged from 5 to 10 mM in strand concentration. The baseline was linearly corrected to account for solvent absorption. Shown in Figure 2a is the FTIR spectrum for a solution of the nitrile tagged single-stranded DNA (ssDNA) oligomers in H2O. The two nitrile groups create two well-resolved bands at EC14N ) 2241.3 cm-1 and EC15N ) 2214.3 cm-1, for the C14NdU and C15NdU bases, respectively. The two bands have equal intensities because the oligomers have equal concentrations and the two nitrile groups are not coupled. This uncoupled system serves as one of two control experiments. Upon addition of the template DNA and annealing by standard protocols, the oligomers associate to form double-stranded DNA (dsDNA) in the B-form. In this geometry, the nitrile labeled bases are stacked on top of one another (Figure 1). The resulting FTIR spectrum (Figure 2b) now exhibits unequal intensities, where the intensity of the lower frequency peak is 1.16 times that of the higher frequency absorption. To further decrease the separation between the two tags, a B- to A-form transition was induced by dissolving the dsDNA construct in an 80%TFE/20%H2O solvent system. In the A-form, the difference in intensities is even more prominent, with the lower frequency peak being 1.66 times taller than the higher frequency peak (Figure 2c). These intensity changes strongly suggest that two nitrile groups are vibrationally coupled. Our first control experiment used single-stranded DNA. To eliminate the possibility that the intensity changes are created by the association of single- into double-stranded DNA, we also performed a second control experiment using double-stranded DNA. In our second control, the nitrile groups are 37 Å apart (and thus uncoupled) in double-stranded DNA. Once again, the intensities are nearly equal (see Supporting Information), indicating that duplex formation does not significantly change the anharmonicity, charge density, or environments of the individual nitrile groups. Thus, the intensity changes observed for closely spaced nitrile groups must be caused by interactions between the nitrile labels. The relative intensities and frequencies of the peaks in each solvent system are related to the coupling and angles between the transition dipoles. The intensities and frequencies are reported in the Supporting Information, obtained by fitting the
J. Phys. Chem. B, Vol. 112, No. 5, 2008 1337 spectra to two Gaussians (or Voigt line shapes in the case of the TFE mixture). The coupling (β) between the two nitrile groups mixes their local modes to create delocalized normal modes. To extract the magnitude of β, we assigned a transition dipole to each nitrile group that is pointed along the bond. The FTIR spectrum was then simulated by diagonalizing a local mode Hamiltonian written in the basis set of the two individual nitrile groups, e.g.,
H)
[
EC14N β EC15N β
]
(1)
and using the resulting eigenvectors to take linear combinations of the local mode transition dipole vectors. The ratio of the squared magnitudes of the resulting normal mode transition dipoles gives the relative FTIR intensities. Since the relative angles of the nitrile groups are known from the canonical DNA structure, the only variable is β. Fits to the intensities give a coupling strength of β ) -1.3 ( 0.3 cm-1 in B-form and -3.9 ( 0.3 cm-1 in A-form DNA. Thus, the coupling strength is very sensitive to the relative distances of the nitriles. The error bars are based on the differences in intensities from the control experiments, which account for inaccuracies associated with DNA concentrations, nitrile labeling efficiencies, background subtraction, and local mode energies, all of which can affect the intensities and, hence, the extracted couplings. Our extracted couplings are based on the intensities and not the frequencies in the FTIR spectra, because the frequency shifts created by the coupling are less than
