<figure><img src="image_1.png" /></figure>

# Chemical Imaging of RNA-Tau Amyloid Fibrils at the Nanoscale
## Using Tip-Enhanced Raman Spectroscopy

Gary Sean Cooney, David Talaga, Vicky Ury-Thiery, Yann Fichou, Yuhan Huang, Sophie Lecomte, and Sébastien Bonhommeau*

**Abstract:** In the presence of cofactors, tau protein can form amyloid deposits in the brain which are implicated in many neurodegenerative disorders. Heparin, lipids, and RNA are used to recreate tau aggregates in vitro from recombinant protein. However, the mechanism of interaction of these cofactors and the interactions between cofactors and tau are poorly understood. Herein, we use tip-enhanced Raman spectroscopy (TERS) to visualize the spatial distribution of adenine, protein secondary structure, and amino acids (arginine, lysine and histidine) in single polyadenosine (polyA)-induced tau fibrils with nanoscale spatial resolution (<10–20 nm). Based on reference unenhanced and surface-enhanced Raman spectra, we show that the polyA anionic cofactor is incorporated in the fibril structure and seems to be superficial to the β-sheet core, but nonetheless enveloped within the random-coiled fuzzy coat. TERS images also prove the colocalization of positively charged arginine, lysine, and histidine amino acids and negatively charged polyA, which constitutes an important step forward to better comprehend the action of RNA cofactors in the mechanism of formation of toxic tau fibrils. TERS appears as a powerful technique for the identification of cofactors in individual tau fibrils and their mode of interaction.

The tubulin-associated unit (tau) protein is an intrinsically disordered protein that binds and stabilizes microtubules (MT) under normal physiological conditions. Under pathological conditions, such as in Alzheimer's disease (AD), the tau protein forms amyloid aggregates found in neurofibrillary tangles. The accumulation of protein inclusions leads to progressive neurodegeneration and global disfunction of brains of affected individuals.[1]

The tau protein contains between 352 and 441 amino acids, depending on the isoform, and is overall positively charged at physiological pH. Tau amyloid fibrils rely on a cross-β-sheet core structure formed by about 60–100 amino acids.[1] Tau filamentous aggregates are hypothesised to form via a nucleation-elongation mechanism, which is promoted by the addition of polyanionic cofactors such as heparin,[2] lipids,[3] and RNA.[4]

These polyanionic cofactors have been shown to be critical to the fibril integrity, with spontaneous depolymerisation and monomer release upon cofactor removal.[2] However, the exact nature of the cofactors incorporated into the mature fibril is still poorly understood, with debate surrounding the role of these cofactors in the formation of pathogenic species as well as their localisation in the mature fibrils.[5–9] RNA is of particular interest as it was shown to colocalize with tau fibrils in AD[10] and to trigger formation of tau fibrils with pathogenic behaviour.[5,7,8] In particular, a recent study suggested that the homonucleotide polyadenosine (polyA) was particularly prone to form tau seeds and amyloid fibrils.[7]

Thanks to the remarkable combination of scanning probe microscopy (SPM) and enhanced Raman spectroscopy, tip-enhanced Raman spectroscopy (TERS) presents the double advantage of high chemical specificity and high lateral spatial resolution, well beyond the diffraction limit. In TERS, the optical excitation of the localized surface plasmon resonance of a noble metal SPM tip induces a strong confinement and enhancement of the electromagnetic field at the tip apex. Moving the resulting hotspot along a sample allows for its nanoscale chemical mapping.[11] TERS has already been used to investigate primary and secondary structure of heparin-induced and phospholipid-induced tau fibrils.[12,13] However, to the best of our knowledge, this study is the first chemical and structural characterisation of RNA-induced tau fibrils with nanoscale spatial resolution.

TERS signatures of biomolecules usually show similar wavenumbers but very different intensities with respect to their unenhanced Raman counterparts.[11,14] Surface-enhanced Raman scattering (SERS) spectra can exhibit intensities closer to those observed in TERS, because they also arise from the resonant optical excitation of plasmonic noble metal nanoparticles at nanometre distance from the probed molecules. However, SERS and TERS spectra may also present differences.[15,16] TERS is indeed more sensitive to changes in molecular orientation than SERS, for which larger sample areas with many hotspots often contribute to the averaged collected signal.[11] Raman and SERS finger-

<figure><img src="image_2.png" /></figure>

<footnote>[*] G. S. Cooney, D. Talaga, Y. Huang, Dr. S. Bonhommeau
University of Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255
33400 Talence (France)
E-mail: sebastien.bonhommeau@u-bordeaux.fr

V. Ury-Thiery, Dr. Y. Fichou, Dr. S. Lecomte
University of Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248
33600 Pessac (France)</footnote>

<footnote>© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
prints thus constitute relevant complementary benchmark
data for TERS spectral assignment of vibrational bands
associated with protein primary and secondary structures.

In this communication, based on the interpretation of
TERS, SERS, and unenhanced Raman spectra, we chemi-
cally and structurally characterise polyA-induced tau (tau-
polyA) fibrils formed from aggregation of monomer units of
the longest tau isoform 2N4R carrying the disease-associated
mutation P301L. Thioflavin T fluorescence assay and trans-
mission electron microscopy confirm the formation of these
fibrils in our experimental conditions (Figure S1). Tau-
polyA fibrils are several micrometres long (Figures S1B and
S1C), 8–13 nm thick and with a helicoidal pitch of 100–
130 nm (Figure S2).

Figure 1 displays the Raman and SERS spectra obtained
for pure polyA, tau-polyA fibrils, and tau-heparin fibrils on
glass and silver-coated glass substrates, respectively (Figur-
es S3 and S4 for more details). A full assignment of Raman
and SERS bands of these spectra is provided in Tables S1–
S3. Nucleic acids are known to adsorb on noble metal
surfaces through the interaction of the nucleoside bases,

rather than the phosphate backbone.[17] The increased
intensity of the Raman band assigned to the backbone
O–P–O stretching modes (790–831 cm⁻¹) with respect to the
corresponding SERS band is likely related to the distance of
this chemical group to the SERS-active nanoparticle surface,
which reduces the SERS enhancement.[18] This distance
dependence of the SERS enhancement originates from the
strong decrease of the electromagnetic field and the loss of
any potential “first layer” chemical enhancement effect with
increased sample-silver nanoparticle distance.[19,20] It suggests
polyA to adsorb to the SERS substrate primarily by the
adenine molecule with the phosphate backbone located at a
greater distance to the surface.

The characteristic adenine ring breathing mode (709–739 cm⁻¹) is used for the unambiguous detection of the presence of polyA within the tau-polyA fibrils as this spectral region is void of protein vibrational modes.[14] While the band corresponding to this mode is intense in the unenhanced Raman and SERS spectra of pure polyA, it is enhanced exclusively in SERS spectra for tau-polyA fibrils (Figure 1). This initial observation suggests the incorporated polyA to be bound to the fibril structure surface in a region accessible to the Ag nanoparticle enhancement. This conclusion is corroborated by a spectral contribution assigned to NH-Ag⁺ (1526–1561 cm⁻¹) in SERS spectra of tau-polyA and pure polyA, but not in the corresponding unenhanced Raman spectra (Figure 1).[21] By comparison, the adenine ring breathing mode is absent in tau-heparin spectra with the heparin contribution observed in the N-sulfate/6-O-sulfate/2-O-sulfate (1032–1065 cm⁻¹) region (Figures 1, S4 and S5).²²

The amide I band (C=O stretching, 1600–1700 cm⁻¹) indicates secondary structure of the protein backbone and is preferential for structural characterization to the amide III band (1200–1340 cm⁻¹) as this mode is sensitive to the dihedral (J) peptide bond angle, and many amino acids contribute within the same spectral region as amide III bands. The unenhanced Raman spectra of both tau-polyA and tau-heparin fibrils have an amide I band with a maximum at 1667 cm⁻¹ (Figure 1). This correlates well with previously reported β-sheet structure of tau-P301L in HEPES buffer at 1671 cm⁻¹ (Table S1).[23] In SERS spectra, the measured amide I bands have maxima correlating to non-regular/random coils at 1634 cm⁻¹ and 1636 cm⁻¹ for the tau-polyA and tau-heparin fibrils, respectively (Figure 1). The highest intensity in the amide I region is not centred at the same wavenumber in Raman and SERS spectra. Considering the decrease of the SERS enhancement with increased distance between the sample and the silver substrate,[19,20] it is thus reasonable to conclude that the disordered “fuzzy coat”, which is expected to be at the closest proximity to silver, is probed in SERS to a greater extent than the core β-sheet structure. This specific amplification of the Raman signal does not occur for disordered secondary structures in unenhanced Raman spectra.

To investigate the tau-polyA fibrillar structure, the
signature of the ring breathing mode of adenine (709–
739 cm⁻¹) and the amide I band secondary structures (β-
sheet, 1660–1682 cm⁻¹; random coils, 1630–1655 cm⁻¹) were

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Raman (dotted lines) and SERS spectra (normalised intensity, full lines) of pure polyA (red), tau-polyA fibrils (green) and tau-heparin fibrils (blue) on glass and silver-coated glass substrates, respectively. Bands corresponding to adenine ring breathing mode and amide I region are indicated as grey bars.</figcaption></figure>
evaluated in 300 spectra of 10 TERS maps of distinct Tau-
polyA fibril portions (Figures 2, S6, S7 and S8). Contrary to
SERS for which the resulting spectra are averaged over an
8.5 µm-diameter sample area with many fibrils, TERS allows
information at the single fibril level to be collected, and
accurate correlations between the protein primary/secon-
dary structure and the presence of polyA cofactor to be
determined with nanoscale spatial resolution.

Figure 2 shows TERS maps performed on two different tau-polyA fibrils with a scanning step size of 10 nm. TERS maps associated with the eight other fibril portions are provided in Figures S6 and S7. The lateral spatial resolution below 20 nm on these maps was consistent with the value reported in a previous study on tau-heparin fibrils in the same experimental conditions (Figure S9).[13] To aid in the visualisation of the colocalization of polyA with secondary structure elements, the TERS dataset was binarized based on a set peak threshold defined as three times the standard deviation of the background noise.[12] This method has been preferred to the usual TERS intensity integration for which intense TERS bands may arise from either favourable molecular orientations or the amount of probed molecular groups (Figure S6). The adenine ring breathing mode (709–739 cm⁻¹) and amide I bands of core β-sheets (1660–1682 cm⁻¹) and of random coils (1630–1655 cm⁻¹) were selected and their binary images produced, reflecting pixels where each of these structures is absent or present (Figures 2Aiii–v and 2Biii–v). These binary images were then combined revealing areas of overlap between adenine and β-sheets (Figures 2Aix and 2Bix), as well as adenine and random coils (Figures 2Ax and 2Bx). PolyA is found to run along the fibril, being colocalised with the core β-sheet when present. Random coils can also be seen in the presence of both adenine and core β-sheets, as well as neighbouring pixels demonstrating the extension of the fuzzy coat from the core.

Considering polyA is negatively charged, we further exploited the TERS maps by looking at three positively charged amino acids, namely arginine (1088–1094, 1156–1171 cm⁻¹), lysine (1070–1080, 1136–1155 cm⁻¹), and histidine (1171–1194, 1327–1337, 1482–1517 cm⁻¹). They are visualized with adenine running along the fibrillar axis (Figures 2Avi–viii, 2Axi–xiii, 2Bvi–viii and 2Bxi–xiii). Due to the complex nature of TERS spectra and protein-associated band overlap, the selected amino acids were analysed using a multiband assignment. For example, it is accepted that a combination of three bands centred at 1180, 1330, and 1495 cm⁻¹ in TERS spectra can be used as a histidine spectral marker.[24] Excluding (but not disregarding) the 6 residue poly-histidine tag of the N-terminus, there are 12 histidine moieties in each native tau protein, with 5 being localised in the R1-R4 microtubule binding domains, 3 in the C-terminus, and the remaining 4 in the N-terminus fuzzy coat.[25] While a quantitative analysis is not possible, Figure 2Bxiii shows the colocalization of histidine with adenine, occurring in regions dominated by both β-sheets (Figures 2Bix and S8B) and random coils (Figures 2Bx and S8B). This suggests our TERS technique is very sensitive to detect both core and fuzzy coat histidine residues. Lysine

and arginine are also assigned using two-band assignment of
terminal ammonium and guanidinium groups, respectively.
Out of the amino acids discernible by TERS, lysine is the
most abundant amino acid in tau with 44 moieties and is
widely distributed throughout the protein. Arginine has 14
moieties with only 1 being present in the R4 and 1 in the C-
terminal domain, with the rest located in the proline rich
domains P1 and P2 and towards the N-terminus. In Fig-
ure S8, lysine and arginine appear abundant in both β-sheet
and random coil rich regions of tau-polyA fibrils. From
Figures 2Axi-xiii and 2Bxi-xiii, it is also clear that adenine is
found in regions primarily with all 3 amino acids, and to a
lesser extent 2 out-of-the 3 amino acids along the fibril core.
This observation supports the hypothesis of anionic cofactor
association with positively charged residues in tau. Other
observable amino acids such as proline, cysteine, tyrosine,
and phenylalanine are not discussed due to their lack of
relevance to the investigated interaction region, their low
TERS enhancement, or their ambiguous assignment due to
band overlap from buffer peaks (Table S3).

Figure 3 presents the proportion of the aforementioned 300 TERS spectra with contributions from adenine (A), random coils (RC), β-sheets (βsh), and their respective combinations. This spectral data set measured using 6 silver-coated TERS probes is large enough to determine reliably the secondary structure of single tau-polyA fibrils with good statistical relevance. As a first note, 4% of all these spectra contained no contribution from either adenine, random coils, or β-sheets, while still having interpretable TERS intensity (above the intensity threshold chosen for binarization in Figure 2). This could be due in part to the suppression of amide I bands which has already been reported with the use of silver TERS probes,[15] and would additionally explain the occurrence of adenine without either random coils or β-sheets (13%). In TERS spectra, amide I bands often appear weak in intensity or can be absent altogether, depending on the direct contact of the amide group and TERS probe[15] provided that no cleavage of peptide backbone bonds occurs.[26] The ultracentrifugation step in the preparation of the fibrils, along with the sample washing after deposition ensures the absence of free unbound polyA. Apart from the 4% of spectra without amide I or adenine bands, 20% of measured spectra contain no adenine contribution. The lack of adenine signal within fibril regions containing either random coils (10%), β-sheets (2%), or both (8%), can have three origins: (1) the absence of polyA in the probed fibril portion, (2) the inconvenient molecular orientation of the pyrimidine ring with respect to the tip axis leading to a weak enhancement, and (3) the burial of polyA within the fibrillar core. The last point is conceivable in principle since TERS probed depth is restricted to a few nm,[13,24,27] and the typical diameter of tau-polyA fibrils falls in the 8–13 nm range (Figures 2 and S2).[6] The total proportion of spectra with random coils (75%) or β-sheets (34%) reflect the typical composition seen in bulk Raman, (random coils (70%), β-sheets (30%)),[23] with differences likely due to the influence of cofactor incorporation (here polyA).
<figure><img src="image_4.png" /><figcaption>**Figure 2.** [A–B] [i] Topographic tapping mode AFM images of distinct tau-polyA fibrils. The height scales are expressed in nm. [ii] TERS spectra associated with one point (white cross in (i)) on the same tau-polyA fibrils. [iii–xiii] Binarized TERS maps obtained from the spectral regions shown as annotated bands in [ii] (100×60 nm, scanning step size: 10 nm, integration time: 5s): [iii] adenine, [iv] core β-sheets, [v] random coils, [vi] arginine (Arg), [vii] lysine (Lys), [viii] histidine (His), [ix] adenine and core β-sheets (yellow), β-sheets only (red), adenine only (green) [x] adenine and random coils (yellow), random coils only (red), adenine only (green) [xi] arginine and adenine (cyan), arginine only (blue), adenine only (green) [xii] lysine and adenine (cyan), lysine only (blue), adenine only (green) [xiii] histidine and adenine (cyan), histidine only (blue), adenine only (green).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3.** [Left] Proportion of 300 TERS spectra across 10 TERS maps that exhibit spectral fingerprints assigned to amide I bands of random coils (RC) or core β-sheets (βsh) and the ring breathing mode of Adenine (A). [Right] Proportional occurrence of adenine with and without tau secondary structures.</figcaption></figure>

PolyA is colocalised with β-sheets at 32% and with random coils at 75% suggesting that it is mostly present in the fuzzy coat. More precisely, it is observed in TERS spectra where random-coiled secondary structures are detected alone (39%), or accompanied with core β-sheet signatures (18%). From this data, we conclude that polyA is not buried deep within the cross-β-sheet network, but rather associates with this network superficially. Given the fluid nature of the “fuzzy coat”, it is likely that this layer shields the core to the TERS probe and only regions of a certain thickness allow the TERS probing of β-sheet structures. Additionally, tau fibrils are helical with a certain rise and fall showing the degree of twist (Figures S1 and S2),[6] with optimal probing of polyA observed in flat regions where the core is effectively probed. The two fibrils analysed in Figure 2 show different patterns for the abundance of adenine with respect to secondary structures, which reflects the topological profile of the tau fibrils (Figures S1 and S2). This explains the high concurrence of random coils with polyA, where all three structures, A + RC + βsh, are simultaneously detected only with relative thinness of the outer fuzzy coat. These findings are supported by the accessibility of the cofactors to digestive enzymes such as RNAse, where the spontaneous depolymerization and release of monomers is shown with the removal of RNA.[5]

Heparin-tau and RNA-tau fibrils formed with recombinant proteins were shown to differ from ex vivo brain-derived fibrils.[5,6,9] While in vivo tau fibrils are believed to be stabilized by a variety of cofactors and post-translational modifications, different anionic cofactors may stabilize particular fibril polymorphs through various sites. The negatively charged phosphate backbone of RNA was believed to promote tau aggregation through interaction with the positively charged β-sheet core. However, recent cryo-EM structures suggest a 36-residue, C-terminal core with bound RNA running parallel to the fibrillar axis that is stabilized via H-bonding between positively charged residues such as arginine, histidine, and asparagine.[6]

Even though we do not discern individual amino acid molecules in TERS, the presence of histidine, arginine, and adenine in core β-sheet regions supports the hypothesis of a C-terminal core bound to polyA running parallel to the fibril axis.[6] The proposed parallel in-register alignment of tau

and polyA could not explain the low intensity of the adenine ring breathing mode observed in TERS, because adenine has a parallel molecular orientation with respect to the tip axis. However, the periodic nature of the helical twist of tau-polyA fibrils leads to regions of weaker and stronger adenine signal along the fibril axis as the TERS tip is periodically closer or further from the polyA bound to the tau protein.

In summary, Raman, SERS, and TERS were used to structurally and chemically characterize tau-polyA fibrils, as prototypical RNA-induced full-length tau protein structures. In these fibrils, the predominance of adenine with both β-sheet and random coils suggests the localization of the anionic cofactor to be superficial to the β-sheet core, but nonetheless enveloped within the fuzzy coat. The accessibility of the cofactor supports the hypothesis of RNA to act as a templating molecule for the building of fibrils.[6] TERS imaging also revealed the colocalization of adenine with positively charged histidine, and arginine moieties implicated in C-terminal core binding, thus confirming specific tau-polyA interactions and providing valuable clues to better understand the efficiency of polyA as aggregation cofactor. Finally, TERS mapping has confirmed that polyA can bind superficially to tau fibrils, forming a unique β-sheet core to that observed with other anionic cofactors such as heparin. This approach opens promising avenues for the characterization of other tau fibrils formed with different cofactors.

## Acknowledgements

All the authors are grateful to the Agence Nationale de la Recherche and the Région Nouvelle Aquitaine for financial support under contract nos. ANR-20-CE29-0004 and 2019-1R1M03, respectively. G.S.C. and S.B. also thank the University of Bordeaux for the MESRI PhD grant for G.S.C., S.B., G.S.C., D.T., and Y.H. thank Sophie Sobanska for her helpful contribution to the instrumental upgrading of the TIR-TERS system. The TERS measurements were performed at the platform SIV (Spectroscopie et Imagerie Vibrationnelle) funded by the European Union (FEDER) and the Région Nouvelle Aquitaine. Y. F. acknowledges the support of the European Research Council (ERC, Grant agreement No. 101040138).

## Conflict of Interest

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.
**Keywords:** Amyloid • RNA • Surface-Enhanced Raman Spectroscopy • Tau Protein • Tip-Enhanced Raman Spectroscopy

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Manuscript received: September 25, 2023

Accepted manuscript online: October 31, 2023

Version of record online: November 13, 2023