SERS and Computational Studies on MicroRNA Chains Adsorbed on

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SERS and Computational Studies on MicroRNA Chains Adsorbed on Silver Surfaces Maurizio Muniz-Miranda,* Cristina Gellini, Marco Pagliai, Massimo Innocenti, Pier Remigio Salvi, and Vincenzo Schettino Dipartimento di Chimica, UniVersita` di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy ReceiVed: April 13, 2010; ReVised Manuscript ReceiVed: July 12, 2010

Surface-enhanced Raman scattering (SERS) of adenine-containing microRNA chains has been obtained by adsorption on roughened silver substrates. The spectral features of all of these samples appear dominated by the bands of adenine. By comparison with the SERS spectra of adenine and adenosine obtained on the same substrates, along with DFT calculations on the interaction sites of adenine and adenosine with silver, inferences are discussed about the structural arrangement of the microRNA chains with respect to the metal surface. This approach gives suitable guidelines in order to investigate the adsorption of complex biomolecules on metal substrates. 1. Introduction The adsorption of biomolecules like nucleic acids and proteins on metal surfaces is the subject of high interest for basic molecular biology1 and for applications in clinical medicine and nanotechnology.2,3 The adsorption processes and the metal/ biomolecule interactions can be studied with the help of vibrational spectroscopy, which provides information on both the dynamic and structural properties of the adsorbates. However, ordinary Raman spectroscopy is usually unable to share these requirements for biological samples, due to low scattering intensity and overlap with fluorescence. These inconveniences can be effectively overcome by resorting to the surface enhanced Raman scattering (SERS) technique,4-6 where huge enhancements of adsorbate Raman signals are coupled with a drastic quenching of fluorescence. As the analyte adheres to surfaces of metals like Ag, Au, and Cu with nanoscale roughness, two mechanisms usually contribute to the Raman enhancement.7 The electromagnetic effect, responsible for enhancement factors higher than 104, arises from the resonance of the exciting and Raman scattering radiations with the plasmon resonance band of the metal electrons localized at the nanostructured surface. The chemical effect, on the other hand, is related to polarizability changes when metal/molecule complexes are formed, producing enhancements up to 102. SERS spectroscopy can reach much higher enhancement factors (1014-1015) in single molecule experiments.8,9 As a consequence, this experimental method combines a fluorescence-like sensitivity with the molecular recognition peculiar to the vibrational spectroscopy. In this context, SERS spectroscopy has emerged as a powerful technique for the characterization of biological materials, in particular single- and double-stranded DNA oligomers, taking advantage of the development and use of reproducible metal substrates.10-12 Following these considerations, in the present study, SERS spectra are reported for microRNA sequences of adenine, adenine/uracil, and adenine/guanine/cytosine (hereafter called AAA, AUA, and AGC, respectively, see Figure 1) adsorbed on roughened silver substrates, and compared with those corresponding to adenine and adenosine adsorbed on both roughened Ag plates and Ag colloidal nanoparticles. The SERS * To whom correspondence should be addressed. E-mail: [email protected].

Figure 1. Chemical structures of adenine and adenosine, along with schematic representations of microRNA chains.

spectra of the microRNA chains as well as that of adenosine are due essentially to the presence of adenine. In order to characterize the molecular site interacting with the metal surface, the adsorbate has been modeled by means of the density functional theory (DFT) as a complex formed by one Ag+ ion and one adenine (or adenosine) molecule. This approach has been successfully proposed by our group for other adsorbed molecules.13-15 The computational results constitute a set of data upon which structural inferences about the microRNA adsorption sites may be worked out. 2. Experimental Section 2.1. Roughening Procedure. Ag plates were polished with successively finer grades of alumina powder down to 0.3 mm (Buehler Micropolish II), mixed with water distilled twice, the first from mineral water, the second with addition of alkaline permanganate to the first purified fraction, discarding heads for both operations. The plates were kept in a stirred solution of 30 mM thiourea (Fluka) and 20 mM Fe(NO3)3 · 9(H2O) for 30 s, producing etching of silver with homogeneous roughness.16,17 2.2. Ligand Adsorption on Roughened Ag Plate. Adenine (Sigma, g99% purity) and adenosine (Sigma, g99% purity)

10.1021/jp103304r  2010 American Chemical Society Published on Web 07/27/2010

SERS and Computational Studies on MicroRNA Chains were used as received. MicroRNA with adenine and uracil (AUA) was purchased from Sigma (purity: desalted); microRNAs (purity: desalted) with only adenine (AAA) and with adenine, guanine, and cytosine (AGC) were kindly provided by Invitrogen srl (Italy). The Ag plates were immersed in 10-3 M adenine or adenosine water solution. After 48 h, the samples were accurately washed with current water and dried at room temperature and the relative SERS spectra were measured, as described in a following subsection. Other roughened Ag plates, freshly prepared according to the chemical treatment abovedescribed, were immersed for 2 days in water solutions of microRNAs (10-5 M concentration) and dried at room temperature. 2.3. Preparation of Ag Colloidal Particles. Silver colloids were prepared following Creighton’s procedure,18 by adding silver nitrate (Aldrich, purity 99.998%) to an aqueous solution of excess sodium borohydride (Aldrich, purity 99%) as a reducing agent. The addition of adenine or adenosine (10-4 M) induced aggregation of the silver nanoparticles, which were deposited and dried on glass to be examined by the microRaman instrument. 2.4. Ag(I)/Adenine Complex Preparation. 5 × 10-2 M AgNO3 aqueous solution was dropwise added to hot 2 × 10-2 M adenine aqueous solution, obtaining a white precipitate, which was accurately washed and filtered at room temperature. The elementary analysis indicated the formation of a 1:1 Ag(I)/ adenine coordination compound with the simplest formula [AgNO3 (adenine) 3 H2O]: Ag weight 30% (expected 30%); N/C weight ratio 1.36 (expected 1.43); H/C weight ratio 0.18 (expected 0.18). The presence of nitrate as a counterion was ascertained in the Raman spectrum by the occurrence of the NO3- symmetric stretching mode at 1044 cm-1. 2.5. Raman Measurements. The SERS spectra of adenine, adenosine, and microRNAs were measured using a Renishaw RM2000 microRaman apparatus, coupled with a diode laser source emitting at 785 nm. Sample irradiation was accomplished using the ×50 microscope objective of a Leica Microscope DMLM. The beam power was ∼3 mW, and the laser spot size was adjusted between 1 and 3 µm. Raman scattering was filtered by a double holographic Notch filters system and collected by an air cooled CCD detector. The acquisition time for each measurement was 10 s. All spectra were calibrated with respect to a silicon wafer at 520 cm-1. The same Raman apparatus was employed to measure the normal Raman spectra of adenine, adenosine, and Ag(I)/adenine complex as crystalline powders. 2.6. Computational Details. DFT calculations have been performed by means of the Gaussian 03 suite of programs19 for adenine, adenosine, and Ag+/adenine model complexes with the Becke three-parameter hybrid functional combined with the LeeYang-Parr correlation functional (B3LYP) and the LanL2DZ basis set. The validity of the computational approach for the Ag+/adenine complexes was verified by the satisfactory simulation of the normal Raman spectrum of adenine, concerning both frequency positions and relative intensities. In particular, the mean error (∑N |νcalc - νobs |/N, where N represents the number of the observed frequencies compared with the calculated ones) is significantly lower than those previously obtained, as shown in Table S1 (Supporting Information). Figure S2 (Supporting Information) shows the satisfactory simulation of the Raman spectrum of adenine, and Figure S3 (Supporting Information) highlights the vibrational assignment of the prominent Raman bands of adenine on the basis of the Cartesian displacements of the corresponding calculated normal modes.

J. Phys. Chem. C, Vol. 114, No. 32, 2010 13731 Also the simulated Raman spectrum of adenosine matches the experimental one, as shown in Figure S4 (Supporting Information), by considering the suitable constraints between adenine and ribose. With two constraints for the ribose ring, the optimized structure is nearly identical to the molecular structure of adenosine in the crystal lattice (see Supporting Information, Figure S5). Analogous DFT calculations (namely, by using the same basis set) were performed also for adenosine interacting with N1, N3, or N7 nitrogen atoms. All structures were optimized with a very tight convergence criterion, and the harmonic frequencies were calculated with an improved grid for the integral calculation, INTEGRAL(GRID ) 199974). The optimized geometries correspond to true energy minima, as revealed by the lack of imaginary values in the calculation of the vibrational modes. The Raman activities (Ai) calculated with the Gaussian 03 program were converted to relative Raman intensities (Ii) using the following relationship derived from the basic theory of Raman scattering20-22

Ii ) f(ν0 - νi)4Ai /νi[1 - exp(-hcνi /kT)]

(1)

where ν0 is the exciting frequency (in cm-1 units), νi is the vibrational frequency (in cm-1 units) of the ith normal mode, h, c, and k are fundamental constants, and f is a suitably chosen common normalization factor for all peak intensities. The calculated spectra were reported by assigning to each normal mode a Lorentzian shape with a 5 cm-1 full width at half-maximum. 3. Results and Discussion 3.1. SERS Results. Ag plates, roughened according to the procedure described in the Experimental Section, are employed for the adsorption of adenine-containing microRNAs, whose sequences are reported in Figure 1. The spectral investigation, performed by microRaman measurements and 785 nm laser excitation, has allowed obtaining suitable SERS spectra from these silver substrates, without significant interference of fluorescence. Also, adenine and adenosine (see Figure 1) are used as ligands, in order to relate their SERS bands to those of the microRNA chains. In Figure 2, the SERS spectrum of adenine adsorbed on a roughened silver plate is compared with that obtained from Ag nanoparticles, exhibiting quite similar spectroscopic features, although with a lower S/N ratio, and indicating the same interaction with silver. The SERS spectra obtained from different substrates, which closely correspond to the normal Raman spectrum (NRS) of the Ag(I)/adenine complex, are dominated by the ring breathing band at 735 cm-1, markedly upshifted with respect to that of NRS of adenine at 722 cm-1. Since adenine in RNA chains is replaced by adenosine, also the SERS spectra of adenosine adsorbed on both a roughened silver plate and Ag nanoparticles have been obtained and compared in Figure 3 with the NRS of solid adenosine. It may be seen that adenosine exhibits a number of bands closely related to those of adenine (722, 1247, 1332, and 1479 cm-1) and, in addition, bands due to vibrational modes more localized on the ribose ring (762, 844, 1507, and 1574 cm-1). The SERS spectra of adenosine from different substrates are quite similar, confirming also for adenosine the same interaction with silver. The strongest SERS band of adenosine occurs at 731 cm-1, corresponding to that observed in the SERS of adenine at 735 cm-1, while the vibrational modes localized on the ribose ring are

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Figure 4. SERS spectra of microRNAs adsorbed on Ag plates. Intensities in arbitrary units. Excitation: 785 nm laser line.

Figure 2. SERS spectra of adenine adsorbed on a Ag plate (C) and on Ag nanoparticles (D), compared with the normal Raman spectra of adenine (A) and Ag(I)/adenine complex (B) as polycrystalline powders. The asterisk refers to the NO3- stretching band of the Ag(I)/adenine complex. Intensities in arbitrary units. Excitation: 785 nm laser line.

Figure 3. SERS spectra of adenosine adsorbed on a Ag plate (B) and on Ag nanoparticles (C), compared with the normal Raman spectrum of adenosine (A) as polycrystalline powder. Intensities in arbitrary units. Excitation: 785 nm laser line.

totally absent. This indicates that adenosine binds to silver in an adsorption arrangement similar to adenine, leading to significant enhancements only for modes localized on the adenine moiety, and suggests that our Raman studies on microRNA samples must be essentially focused on the detection of the marker band at 731 cm-1. In fact, the SERS spectra of all three microRNAs (Figure 4) are dominated by the 731 cm-1 band, so that the chains are reasonably bound to the Ag substrate through this nucleobase. In the SERS spectrum of AAA, other bands occur at 630, 1267, 1330, and 1460 cm-1, in close proximity to those of adsorbed adenine (see Figure 2). For the AUA chain, where adenine and uracil nucleotides alternate, additional weak bands

are observed at 790 and 1235 cm-1, whereas, for the AGC chain, where adenine and guanine are present with a smaller percent of cytosine, a further weak peak is found at 675 cm-1. Comparison with reported data11,23 suggests for the latter bands of AUA and AGC an assignment to Raman modes of uracil and guanine, respectively. In saturated water solution of uracil at neutral pH and for the polycrystalline powder, two intense peaks, 784/790 cm-1 and 1235/1236 cm-1, occur in the NRS.23 Therefore, the AUA peaks, located at 790 and 1235 cm-1, are attributable to uracil not directly bound to the substrate, confirming that the chain interacts with the substrate through adenine. Further, in agreement with the assignment of the breathing band of guanine at 678 cm-1 in the NRS of the SA20N2 oligonucleotide sequence (containing guanine),11 the same assignment is here proposed for the 675 cm-1 SERS band of AGC. The absence of spectral features related to cytosine24 in the SERS spectrum of AGC agrees with the fact that this nucleobase is a minor component of the chain not directly involved in the chemisorption. 3.3. MicroRNA Interaction Sites. The adsorption of adenine on metal surfaces has been discussed in much detail over the years;25-35 in particular, this nucleobase was reported to adsorb on silver surfaces Via different nitrogen atoms of the two rings (N1, N3, and N7 atoms) or through the external amino group, in either a side-on or a flat-on orientation. Undoubtedly, these controversial conclusions were related to the variety of interaction sites and to the different tautomeric forms of this molecule. In fact, adenine is known to exist in two forms in solution, with the 9-H tautomer dominant over the 7-H tautomer.36-38 On the other hand, DFT calculations on surface complexes with adenine bound to one silver atom suggested an interaction through the N3 nitrogen,32 consistent also with that recently proposed for adenine on gold.35 The authors of ref 31, however, recognized that other model systems involving silver clusters or silver ions could provide a more precise picture of the SERS spectrum concerning both frequency shifts and relative intensities. Here, the silver/adenine interaction geometry is examined mainly in relation to the structural changes possibly occurring when adenine enters as a component of the microRNA sequences. In this respect, the Raman spectrum of the Ag(I)/ adenine complex (Figure 2B) appears almost identical to the SERS of adenine (Figure 2C), apart from the symmetric stretching band of the NO3- counterion at 1044 cm-1, indicating that the interaction of adenine with the silver surface resembles that of the Ag(I) coordination compound. This point provides useful information: (i) adenine interacts with silver as a neutral

SERS and Computational Studies on MicroRNA Chains

Figure 5. Optimized structures of Ag+/adenine complexes obtained by means of DFT calculations.

TABLE 1: Calculated Energies, ∆E, in cm-1 and kJ · mol-1 of Ag+/Adenine Complexes Shown in Figure 5 with Respect to the Lowest Energy Complex (d)a ∆E model complex cm

-1

kJ · mol-1 νbreathb ∆νbreathc

a1 a2

2092 3862

25.03 46.20

712 713

2 3

b c1 c2

2057 3862 2678

24.61 46.20 32.04

728 714 701

18 3 -9

725

15

710 714

0 4

d e1 e2

6741 7569

80.64 90.55

r(Ag · · · Nx) r(Ag · · · N1) ) 2.167 r(Ag · · · N1) ) 2.324 r(Ag · · · N10) ) 2.468 r(Ag · · · N3) ) 2.158 r(Ag · · · N7) ) 2.156 r(Ag · · · N7) ) 2.304 r(Ag · · · N10) ) 2.400 r(Ag · · · N3) ) 2.223 r(Ag · · · N9) ) 2.808 r(Ag · · · N1) ) 2.171 r(Ag · · · N1) ) 2.264 r(Ag · · · N10) ) 2.582

a The corresponding breathing mode frequencies, νbreath (cm-1), the frequency shifts with respect to that of adenine, ∆νbreath (cm-1), and the distances between the Ag+ ion and the closer nitrogen atoms of adenine, r(Ag · · · Nx) (Å), are reported. b The breathing mode frequency of adenine is 710 cm-1, according to our DFT calculations. c The experimental shift of the SERS mode of adenine adsorbed on a silver plate with respect to the corresponding Raman mode is 13 cm-1.

molecule, not in anionic form; (ii) the adsorption of adenine on silver can be suitably modeled like a molecule bound to one silver ion, as in the Ag(I) coordination compound. For this reason, the present DFT calculations have been performed on adenine molecule and on Ag+/adenine complexes with one silver ion linked alternatively to N1, N3, or N7. Eight minima structures, a1, a2, b, c1, c2, d, e1, and e2, have been found (Figure 5), by considering adenine also in the 7-H tautomeric form. The energies of the different Ag+/adenine complexes are reported in Table 1. Not surprisingly, the lowest minimum corresponds to the tautomeric structure d, where the molecule is bound to the silver ion through N3 and (in a weaker way) N9. The second most stable structures are a1 and b, where the molecule interacts with silver Via one nitrogen atom of the pyrimidinic ring. The Ag+/adenine complex involving the amino

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Figure 6. Cartesian displacements of the calculated breathing modes of Ag+/adenine complexes with interaction sites located at N1, N3, and N7, corresponding to the SERS band of adenine observed at 735 cm-1. Hydrogen atoms are omitted for the sake of simplicity.

group has been excluded from our calculations, since the structure migrates toward a1 during the optimization step. Considering now the vibrational results, the most striking experimental difference between NRS and SERS of adenine is relative to the ring breathing mode: the NRS band at 722 cm-1 undergoes a significant upshift, ∼13 cm-1, when adenine is adsorbed on silver, along with a marked increase of the relative intensity, as shown in Figure 2. The calculated upshifts for the b and d complexes are 18 and 15 cm-1, respectively. The basic indication from the vibrational calculations is therefore that the breathing mode frequency considerably upshifts only when Ag+ is bound to N3. The calculated frequency shifts of Table 1 make this point particularly evident. Moreover, by considering the Cartesian displacements of this mode in the N1, N3, or N7 complexes (Figure 6), the nitrogen atom bound to silver moves significantly from the equilibrium geometry toward the metal only in the b complex. A similar behavior has been observed for the d structure. This justifies the intensity increase of the corresponding SERS band on the basis of the surface selection rules,38 for which totally symmetric vibrations showing large shifts of the equilibrium position in the direction normal to the metal surface should be greatly enhanced. In fact, for an edgeon adsorption through N3, the motion of the nitrogen atom with its electronic density induces a large polarizability change along the direction normal to the silver surface and, consequently, strongly enhances the SERS band. The SERS bands observed at higher frequencies (1273, 1331, 1372, and 1459 cm-1) correspond, instead, to normal modes not appreciably involving the N3 motion. In conclusion, the DFT calculations of the Ag+/ adenine complexes point to an interaction of adenine with silver Via N3, described by either b or d geometries. The SERS frequencies are satisfactorily calculated by means of both model systems, as shown in Table 2. The d model, where adenine interacts with silver in the 7-H tautomeric form, is more plausible on the basis of the strong stabilization of this complex (Table 1) and the good matching of the simulated SERS profile with the experimental one, as shown in Figure 7. Actually, the ring breathing mode in the Ag+ complex appears strongly enhanced with respect to the corresponding mode of the free molecule, as experimentally observed.

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TABLE 2: Experimental SERS Frequencies (cm-1) of Adenine, Adenosine, and AAA MicroRNA Compared with Those Calculated (cm-1) for the b and d Complex Models SERS

calculated

adenine Ag plate

adenosine Ag plate

AAA Ag plate

b model

d model

Sa

assignment

1459 1372 1331 1273 735 632 570

1460 1365 1328 1269 731 628 566

1460

1466 1397 1354 1250 728 610 543

1470 1384 1346 1253 725 600 553

A′ A′ A′ A′ A′ A′ A′

ring stretching + H bending H bending + ring stretching H bending + ring stretching H bending + ring stretching ring breathing ring bending ring bending

a

1330 1267 731 630

Symmetry species (Cs group).

Figure 7. Simulated SERS spectrum (B) of adenine (d complex model), compared with the simulated normal Raman spectrum (A) and with the observed SERS spectrum (C).

The b and d structures can be used as a basis for the discussion on the interaction sites of microRNAs, whose SERS spectra are dominated by the adenine bands (see Figure 4). On the other hand, since in microRNAs the N9 site is occupied by a ribose group, as well as in adenosine, the preferred adsorption structure of adenine in these nucleobase chains corresponds to model b, by interaction with silver only through the N3 nitrogen. To verify this conclusion, DFT calculations have been performed on adenosine linked to metal via N1, N3, or N7. In spite of the simplicity of the model systems, where the metal surface is modeled by one silver ion, the simulated SERS profiles legitimate the indication of N3 as the most probable interaction site. As shown in Figure 8, only the Ag+/adenosine complex with N3 interaction is able to satisfactorily reproduce the observed strong enhancement of the ring breathing mode observed at 731 cm-1. The dominance of the adenine bands, observed in the SERS spectra of microRNAs, was previously observed in the SERS of adenine-containing DNA oligomers adsorbed on Au substrates and attributed to the higher SERS cross section of this nucleobase.11 In the present study, the interaction of microRNAs to silver Via the N3 nitrogen atom of adenine can be reasonably proposed on the basis of the SERS and DFT results for adenine and adenosine. This fact is consistent with a stronger chemical interaction of the adenine nucleobase with the metal surface, because the pyrimidinic ring is directly involved in the bond with silver. Unlike other DNA/RNA nucleobases (thymine,

Figure 8. Simulated SERS spectra of adenosine by interaction of the N1, N3, or N7 nitrogen atom with one silver ion, compared with the SERS spectrum of adenosine (upper panel).

uracil, guanine, cytosine), the pyrimidinic ring of adenine has aromatic character, favoring a stronger metal/molecule chemical interaction and, as a consequence, a stronger SERS enhancement. In this respect, it is interesting to note that the quasiisoenergetic a1 and b structures (Table 1) describe the possible interactions of adenine by means of the N1 or N3 nitrogen atoms of the pyrimidinic ring, which are more efficient than those involving the N7 nitrogen atom of the five-membered ring. This displays the importance of involving the delocalized π electrons of the aromatic ring in the chemical bond with silver, which could be used as a useful guideline for investigating the adsorption processes of these biomolecules. Finally, some qualitative considerations can be advanced about the binding of adenine to the silver substrate in the microRNA chains. Reference is made to AAA oligomers for which several sets of structural data have been reported.40-44 It is in fact known that oligoAAAs exist in single-strand conformations40,41 and polyA (polyriboadenylic acid) as a singlestrand flexible helix,42 both structures being stabilized by base stacking. A model has been described for polyA in more detail42 as a single helix with a pitch height of ∼25 Å and nine

SERS and Computational Studies on MicroRNA Chains nucleotides per turn, on the basis of the structure of the trinucleoside diphosphate ApApA.44 A similar helicoidal arrangement can be reasonably proposed for the microRNA chains. The occurrence of the ring breathing mode of adenine at the same frequency (731 cm-1) for all microRNAs suggests that the same base is involved in the interaction with the metal, probably the initial adenine in the nucleobase sequences. 4. Conclusions SERS-active silver substrates have been prepared by chemical roughening and employed for the adsorption of adenine, adenosine, and adenine-containing microRNA chains. These SERS platforms, which provide quite similar spectroscopic findings in comparison with the silver colloidal nanoparticles, are easy to fabricate, stable in time, and reusable after successive surface treatment. Moreover, the adsorption process merely consists of plunging a roughened silver plate into the ligand solution, which may then be retrieved. These advantages, with respect to other SERS substrates as silver colloids, widely compensate a lower S/N ratio. They, however, exhibit quite satisfactory SERS efficiency: an approximate evaluation of the effective quantity of ligand observed by a microRaman measurement, by comparing the UV absorption of the solution before and after adsorption on silver, results for AAA microRNA in a very small amount, ∼10-17 mol. The SERS spectra of microRNAs, where the bands of adenine appear dominant, have been analyzed by comparison with those of adenine, adenosine, and the Ag(I)/adenine coordination compound. A detailed DFT study on the possible silver/adenine surface complexes has been performed. This investigation, which points to an interaction of adenine in the 7-H tautomeric form with the silver surface through the N3 nitrogen atom of the pyrimidinic ring, sheds a new light on the “vexata quaestio” concerning the adsorption of adenine on metal. Our conclusions are based not only on the stability of the surface complexes and the agreement between calculated and observed wavenumbers but also on the very satisfactory simulation of both the frequency shift and the intensity increase for the marker SERS band of adenine observed around 730 cm-1. The present study allows also suggesting reliable considerations about the adsorption on metal and the structural arrangement of complex systems like RNA/DNA polynucleotide chains, along with an acceptable explanation for the predominance of the adenine bands in the SERS spectra of these biomolecules, essentially based on the involvement of aromatic π electrons in the interaction with the metal surface. Acknowledgment. The authors gratefully thank the Italian Ministero dell’Universita` e Ricerca for the financial support and Dr. Barry Howes (University of Firenze), who kindly provided us microRNA samples. Supporting Information Available: Table showing vibrational frequencies and figures showing Raman spectra, Cartesian displacements, and optimized structures. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Davis, L. G. Basic Methods in Molecular Biology; Appleton & Lange: Noralk, CT, 1994. (2) Pierstorff, E.; Ho, D. J. Nanosci. Nanotechnol. 2007, 7, 2940. (3) Herne, T. M.; Ahern, A.; Garrell, R. L. J. Am. Chem. Soc. 1991, 113, 846. (4) Chang, R. K.; Furtak, T. E. Surface-Enhanced Raman Scattering; Plenum Press: New York, 1981. (5) Otto, A. In Light Scattering in Solids; Cardona, M., Gu¨ntherodt, G., Eds.; Springer-Verlag: Berlin, 1984; Vol. IV, p 289.

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