Tetracationic Bis‐Triarylborane 1,3‐Butadiyne as a Combined Fluorimetric and Raman Probe for Simultaneous and Selective Sensing of Various DNA, RNA, and Proteins

Abstract A bis‐triarylborane tetracation (4‐Ar2B‐3,5‐Me2C6H2)‐C≡C−C≡C‐(3,5‐Me2C6H2‐4‐BAr2 [Ar=(2,6‐Me2‐4‐NMe3‐C6H2)+] (24+) shows distinctly different behaviour in its fluorimetric response than that of our recently published bis‐triarylborane 5‐(4‐Ar2B‐3,5‐Me2C6H2)‐2,2′‐(C4H2S)2–5′‐(3,5‐Me2C6H2‐4‐BAr2) (34+). Single‐crystal X‐ray diffraction data on the neutral bis‐triarylborane precursor 2  N confirm its rod‐like dumbbell structure, which is shown to be important for DNA/RNA targeting and also for BSA protein binding. Fluorimetric titrations with DNA/RNA/BSA revealed the very strong affinity of 24+ and indicated the importance of the properties of the linker connecting the two triarylboranes. Using the butadiyne rather than a bithiophene linker resulted in an opposite emission effect (quenching vs. enhancement), and 24+ bound to BSA 100 times stronger than 34+. Moreover, 24+ interacted strongly with ss‐RNA, and circular dichroism (CD) results suggest ss‐RNA chain‐wrapping around the rod‐like bis‐triarylborane dumbbell structure like a thread around a spindle, a very unusual mode of binding of ss‐RNA with small molecules. Furthermore, 24+ yielded strong Raman/SERS signals, allowing DNA or protein detection at ca. 10 nm concentrations. The above observations, combined with low cytotoxicity, efficient human cell uptake and organelle‐selective accumulation make such compounds intriguing novel lead structures for bio‐oriented, dual fluorescence/Raman‐based applications.


Introduction
The use of fluorescentl abels to image specific small molecules in live cellsi sa ne ssential tool in biology,m edicinal chemistry, and many other related fields of research. However,c ommon fluorescent dyes also have many drawbacks.D ue to the considerable size and aromaticn ature, and also often being H-bond donors/acceptors, they may alter the biological activity,c ellular localization, and dynamics of the targeted bio-molecules. [1] Moreover,e ven the most advanced fluorescencem icroscopy techniques are limited to am aximum simultaneous resolution of six different colors. [2] This is additionally complicated [3] by unavoidable cross-talk in organic dyes, energy transfer between quantumd ots [4] and the limited number of suitable features for straightforwardd ecoding in rare-earth nanocrystalsand metal nanoparticles. [5] Therefore, furtherd evelopment in the increasingly complex bioimaging fielde ssentially requires newf luorophore structures for better bio-target diversification. Even more useful would be new optical probes based on intrinsically different methodsa tl east approachings imilar sensitivities of common fluorophores. Such an aim recently led to the development of vibrational spectroscopym ethods, particularly Ramanb ioimaging microscopy,d ue to its compatibility with aqueous biological samples. [2d] Phenyl end-capped polyynes have been employed as alkyne tags for Raman visualization of mobile, small molecules in cells [6] as well as for surface-enhanced Raman spectroscopy (SERS)m ultiplexc ellular imaging, [7] showing very promising applications in supermultiplexed optical imaging and barcoding. [2d] However,t hese polyyne-probesr eliede xclusively on their Ramanr esponse, and thus were not combined with other sensing techniques.
Althoughf luorescence, under certain experimental conditions, interferes with Raman measurements, by causing significant background signal, [8] for some chromophores it is possible to obtain as atisfying compromise. In recent years, the combination of Ramana nd fluorescences pectroscopyh as emerged as aw ay to circumvents ome of the intrinsic problemso f Raman spectroscopy (i.e. low signal strength,l ong acquisition times) or to gain additional information on ag iven system. [9] Thus, in the first multimodal approaches,f luorescencew as used as af ast macroscopic scanning method, prior to ad etailed Ramana nalysis, or to confirmc onclusions made via Raman-based imaging. [10] Ramans pectroscopy, in combination with the use of fluorescently labelled molecules or quantum dots, also proved to be an interesting approachi ns ome cell imaging applications. [11] In disease diagnostics, dual Raman and fluorescences pectroscopy was used by severalg roups in a complementary fashion to improve the accuracy and sensitivity of the diagnosis. [12] Dual fluorescence and Raman spectroscopy was successfully employed to investigate and visualize intracellular drug delivery. [13] The design of small molecules with inherently strongR amana nd fluorescencer esponses is ar ather novel approach, [14] which has already produced much information on the location, environment [14b] or concentration [14a] of the small molecule inside acell.
Recently,t riarylboranes have emerged as as tructurally novel class of compounds suitable for biological imaging applications. [15] Over the last decades, triarylboranes have also found applicationsi nm any other fields, such as anion sensors, OLEDs and non-linear optical materials. [16] Due to its vacant p z -orbital, the three-coordinate boron in at riarylborane is as trong p-acceptor.However,itisalso Lewis-acidic and sensitivetohydrolysis. Bulky substituents can stabilizet hree-coordinate boron against decomposition by air and moisture, while maintaining its p-acceptor strength. [17] Using Gabbaï's approach [18] we developedt he water-soluble, water-stable and non-cytotoxic tetracationic chromophore 3 4 + + (Scheme 1) whichw as successfully utilized in live cell imaging. [19] Further studies revealed 3 4 + + to be as tructurally novel DNA/RNA/protein probe of high affinity and selective fluorimetric and chirooptic response. [20] The question arose to which extent the chromophoric linker (bithiophene) is responsible for the observed spectroscopic responses and affinity.I nas earch for linkersw hich could also be non-conventional response probes, 1,3-butadiyne attracted our attentionf or several reasons. It is ar igid, rod-like symmetrical linker,e xcellent for precise orientation of terminal triarylborane cations (Scheme 1, 2 4 + + ), and it is completely inert, in terms of covalenta nd non-covalent interactions, to DNA/RNA/protein. Even more importantly,i te xhibits an intense Ramanb and with an arrow linewidthi nt he Raman-silent spectral region (2250-2000 cm À1 )c onvenientf or Raman-based probingi na queous solutions. Additionally,t he intensity of response can be strongly increased by surface-enhanced Ramans cattering (SERS) spectroscopy and relatedt echniques.
Thus, novel molecule 2 4 + + ,a nd studies of its interactions with DNA/RNA/proteins, will address the potentialf or the development of innovative combined fluorophore-Raman probes for the general purpose of non-covalent probing of DNA/RNA/ proteins,a nd the impact of the nature of the linker connecting two bis-triarylborane cations on thef luorescent and chirooptic response upon bindingt ob iomacromolecules.
Given the generally weaks pontaneous Raman responseo f molecules in solution, SERS spectroscopy was used to study the bindingt ob iomoleculesi na queous media mimicking the biological environment. The enhancement of the Raman scattering in the vicinity of nanostructured metal surfaces arises from amplification of the electromagnetic field upon excitation of the localized surface plasmon resonances (LSPR) of the molecules when physisorbedo nasurface, and from the transfer of electrons from the metal to the molecule and vice versa for chemisorbed molecules. [21] Even though the charge transfer mechanism is considered to contributet ot he overall enhancement to al esser extentthan the electromagnetic one, the total SERS enhancementf actor with respectt ot he normalR aman signal, in most cases, is the product of both mechanisms, reachingu pt o1 0. [10] Owing to its high sensitivity and ability to produce molecularly specific fingerprint spectra, SERS has been successfully appliedf or the detection, quantification and biophysicalcharacterization of avariety of biomolecules. [22] Results and Discussion
Single crystals suitable for X-ray diffractiona nalysisw ere obtained for compounds 1N ( Figure S17) and 2N.T he solid state molecular structure of 2N,a long with selected bond lengths and angles, are depicted in Figure 1, to illustrate the shape and size of the diyne compound.T he distance between the two boron atoms B1 and B1' is 15.457(7) .T he molecule has an inversion center located between C1 and C1' and is almost linear,a st he relevant CÀCÀCb ond angles of the diyne bridge are all closeto1 808.

Physicochemical properties
Both positively charged compounds (1 2 + + , 2 4 + + )a re moderately soluble in water (c = 1 10 À3 m), their solutions being stable for longerp eriodsw hen stored in the dark. The neutral analogue of 1N was dissolved in DMSO to prepareastock solution (c = 5 10 À3 m)w hich was further diluted with aqueous buffer prior to every experiment,y ieldings The absorbances of the studied compounds ( Figure 2) were proportional to their concentrationsu pt oc = 2 10 À5 m (Figures S21-S23), and changes in their UV/Vis absorption spectra upon temperature increase up to 90 8Cw ere minor (Figure S24). Reproducibility of the UV/Vis spectrau pon cooling to 25 8Cw as excellent. These findings indicate that the compoundsd on ot aggregate by intermoleculars tacking under the experimental conditions used. Absorption maxima and corresponding molare xtinction coefficients (e)a re given in Ta ble 1.
Comparison of their UV/Vis absorption spectra reveals distinct differences betweent he neutral compound 1N and its charged analogue 1 2 + + .T he neutral molecule 1N shows an absorptionb and at 408 nm while the charged analogue 1 2 + + shows ah ypsochromically shifted absorption with am aximum at 315 nm and as houldera t3 35 nm. This absorption behavior is well documented in previous publications from our group for as eries of analogousn eutral and charged compounds [17p, 19, 26] and can be attributed to al oss of the charge transfer transition from the amine to the boron moietyu pon methylation of the amine. The approximate doubling of the extinction coefficienta nd the bathochromic shift of > 40 nm (3300 cm À1 )i na bsorption, when comparing the monomer 1 2 + + and the dimer 2 4 + + ,i sa lso consistent with our previous studies [17p, 19] and can be attributed to al arger p-systemi nt he case of 2 4 + + .All photophysical data is summarized in Ta ble 1.

Study of interactions with DNA, RNA, and BSA
To study the interactions of 1 with DNA/RNA, severalt ypical types of DNA and RNA were chosen (Table S2). Naturally occurring calf thymus (ct)-DNA represents at ypical B-helix structure with ab alanced ratio of GC-(48 %) and AT-(52 %) base pairs. Synthetic alternating polynucleotides poly (dGdC) 2 and poly (dAdT) 2 represent two extreme situations (only AT-o rG C-basepairs, respectively), differing significantly in their secondary structures as well as in the availability of the minor groove for small molecule binding( the guaninea mino group sterically   hinders deep molecule penetration). For comparison between double stranded (ds) DNA and ds-RNA, poly rA -p oly rU RNA was chosen as an A-helix structure characterizedb yamajor groove availablef or the bindingo fb ulky small molecules.
Furthermore,t oe xplore the DNA/RNA binding of the novel chromophore to ag reater extent, we also studied the single stranded synthetic ss-RNA polynucleotides poly G, poly A, poly Ua nd poly C, each of them characterizedb yd ifferent properties. Adenine ss-RNA mimics 50 to 250 adeninen ucleotides at the 3' end of mRNA,p oly Gi sr elatedt og uanine-rich sequences in both DNA and RNA, whereas poly Ca nd poly Ua re significantly more flexible than purine-RNAs, and with less organized secondary structures.
Due to the possibility that the compounds studied could interact with proteins, we examined the most naturallyabundant protein, bovine serum albumin (BSA), taking into account its versatility of binding sites.

Thermal denaturationexperiments
It is well known that double stranded (ds)-helices of polynucleotides dissociate into two single stranded polynucleotides upon heating at well-definedt emperatures (Tm value).Non-covalent binding of small molecules to ds-polynucleotidesusually increases the thermal stability of the ds-helices thus resulting in increased Tm values, and the increase (DTm)c an (corroborated by other methods) be related to the various binding modes. [27] Te tra-charged 2 4 + + stabilized ds-DNA moderately (r [ Furthermore, 3 4 + + showed an emission increaseu pon DNA/ RNA/BSA complexation,c haracterized by distinct differences in fluorescencem axima between DNA/RNA and protein (BSA), whereas 2 4 + + showed only non-selectivee mission quenching. Such ad ifferencei nf luorimetric response between 2 4 + + and 3 4 + + might be attributedt ot he fact that bithiophenei saweak donor,w hereas the diyne unit does not have an electron-donating effect.
The non-linear fitting of the DNA or RNA titration data by means of the Scatcharde quation (McGhee, vonHippel formalism) [28,29] allowed the calculation of binding constants ( Table 2). The BSA titration data fitted excellently to a1 :1 (2 4 + + :BSA) stoichiometry model, pointing to only one dominant binding site of 2 4 + + at BSA.
The strong, submicromolar affinities of 2 4 + + to ds-DNAa nd ds-RNA were within the same ordero fm agnitude, while its affinity to ss-RNAw as approximately an order of magnitude lower.T he affinity of 2 4 + + to BSA was an ordero fm agnitude higher than its affinity towards ds-DNA/RNA ( Table 2). The excellent fit of the titration data ( Figure S42) strongly supported as ingle dominant binding site on BSA for 2 4 + + ,a lthough other binding sites with several orders of magnitude lower affinities cannotb ee xcluded. Comparison of the affinities between 2 4 + + and bithiophene analogue 3 4 + + revealed similar binding constants for ds-DNA/RNA and ss-RNA, but as ignificant difference in binding to BSA, with 2 4 + + showing an affinity two orders of magnitude higher than 3 4 + + .
Monomer 1 2 + + revealed somewhat lower affinity to ds-DNA/ RNA, but still in the micromolar range,a lbeit having only half of the positive charge, suggesting that electrostatic interactions with the negatively charged DNA backbone are not the dominant binding interactions. Intriguingly,t he emission of 1 2 + + did not change with BSA or ss-RNA addition, indicating that the long rod-like structure of dimer 2 4 + + is essential for efficient binding to both targets.

CD experiments
Thus far,w eh ad studied the non-covalenti nteractions at 25 8C by monitoring the spectroscopic properties of the compounds upon addition of the polynucleotides. In order to obtain in-sight into the changes of polynucleotidep roperties induced by small molecule binding, we chose CD spectroscopy as a highly sensitivem ethod for the examination of conformational changes in the secondary structure of polynucleotides. [30] In addition, 1 2 + + or 2 4 + + as achiral small molecules could display induced circulard ichroism (ICD) within their absorption spectra upon binding to polynucleotides, which could provide useful information about modes of interaction. [31,32] Addition of 1 2 + + did not significantly change the CD spectra of ds-DNA or ds-RNA ( Figures S44-S46), and no induced (I)CD bands > 300 nm were observed. In contrast, tetracation 2 4 + + induced as ignificant decrease in intensity in the CD spectra of all ds-DNA/RNA (230-300 nm range;F igure 5), attributed to a pronounced decreaseinds-polynucleotide chirality. [31,32] Only poly (dAdT) 2 ( Figure 5) revealed significant induced (I)CD bands at l > 300 nm, which could be attributed to the uniformly oriented binding of 2 4 + + within aw ell-defined DNA binding site. [32] Taking into account the structure of the molecule 2 4 + + ,t he DNA minor groove is the most plausible binding site. Closer inspection of the ICD bands and comparison with the UV/Vis titrationd ata ( Figure 5, Inset:b lack line) revealed a mixed binding mode of 2 4 + + ,d ependent on ar atio r [compound]/[poly (dAdT)2] ;w hereby for r < 0.2 ICD bands were negative and for r ! 0.3 ICD bands were positive.S uch ac hange of the ICD sign is commonly attributed to singlem olecule binding at an excesso fD NA (r < 0.2) and molecular aggregation within the DNA grooves at an excess of the small molecule (r > 0.3). [31,32] Further,d ominant ICD bands at 300-330nmc ould be partially attributedt oe lectronic transition vectors along the boron-nitrogen axes (see Ta ble S5), which seem to be well oriented with respect to the DNA chiral axis. The maxima at l max = 360-380 nm), giving negligible ICD band, were attributed to the electronic transition along the long axis of 2 4 + + ,parallel to the diyne-linker (see Ta bleS5).
The almost negligible intensity of ICD bands for the analogue 2 4 + + /GC-DNAc omplex could be attributed to the sterically crowded minor groove with the amino groups of guanine, not allowing deep insertion of 2 4 + + and, thus, diminishing the induced CD effect. [32] The broad ands hallow minor groove of AU-RNAi sapoor binding site for small molecules,i nc ontrast to the major groove of RNA,w hich has aw idth similar to that of the minor groove of DNA (Table S2) and could be an efficient binding site for 2 4 + + .H owever, the large depth of the major groove allows heterogeneous orientationo f2 4 + + molecules with respect to the ds-RNA chiral axis, thus resulting in negligible ICD bands.
Particularly intriguing results were obtained for the 2 4 + + /ss-RNA complexes ( Figure 6). Addition of 2 4 + + completely disordered the CD spectrum of poly Aa nd also that of poly U, while the CD spectra of poly Ga nd poly Cw ere lessa ffected. For the poly At itration,t he isoelliptic point at l = 253 nm strongly supported only one type of 2 4 + + /poly Ac omplex. The poly Ut itration showed as ystematic shift of the spectralc rossing points during titration,w hich is typical for mixed binding modes.
Complete loss of the helical chirality of Ao rUss-RNA upon binding to 2 4 + + ,a ccompanied with ar ather high affinity of 2 4 + + [a] Analyses of titration data by means of the Scatcharde quation [28,29] gave valueso ft he ratio n    Table 2) suggested wrapping of the ss-polynucleotide chain aroundthe cylindrically shaped compound 2 4 + + .Such abinding mode would maximize the efficiency of electrostatic interactions betweenf our positivec harges of 2 4 + + and the negative polynucleotideb ackbone. This binding mode would be additionally supported by an energetically favorable exclusion of the hydrophobic diyne linker from hydrophilic solvent molecules. Such ac omplex of achiral 2 4 + + serving as as pindle for ss-RNA would not give anyc hiral response, which is in accordance with the CD titration experiments ( Figure 6).
Another proof of the proposed binding mode is that monomer 1 2 + + did not changet he CD spectrum of ss-RNA, which could be attributed to the globulars hape of 1 2 + + with its centered positive charge, not supporting ss-polynucleotide wraparound.

Raman spectroscopy
Raman spectra of 2 4 + + were measured in water (1 10 À4 m)a nd Na-cacodylate buffer, (pH 7.0, 1 10 À4 m and 5 10 À5 m) (Figure 7a nd Ta ble S4).I nt he Raman spectra of all solutions, a broad band around 3220 cm À1 and am edium band around 1640 cm À1 were observed and assigned to the water stretching and bending modes, respectively.I na ddition, in the Raman spectra of the buffered solutions, bands originating from cacodylate ions wereo btained:amedium methyl asymmetric stretching band at 2935 cm À1 ,aweak methyl deformation band at 1414 cm À1 and an intense As=Os tretching band at 605 cm À1 .Nevertheless, bands distinctiveo f2 4 + + were observed in all Raman spectra, even at ac oncentration as low as 5 10 À5 m (Table S4). Hence, the band around 2220 cm À1 was assigned to the stretching of the CCt riple bondsi nb isarylsubstituted diynes, [6] while the band around 1600 cm À1 ,o verlapped by the water band, was attributed to ap henyl ring stretching mode (Table S4). [33] Based on the calculated Raman spectrum of 2 4 + + (Figure S49), the band around 1355 cm À1 was associated with mixed stretching vibrations of thet riple bonds and phenyl rings, whereas stretching of the bonds between the boron atom and three aromatic substituents contributed to the band around 1070 cm À1 .T he strong Ramans cattering of 2 4 + + was attributed to the diyne moiety conjugatedt ot he aromatic rings, [34] allowing detection of the molecules in solution at amicromolar concentrationr ange.

SERS experiments
The SERS spectra of 2 4 + + were measured in the 5 10 À8 -5 10 À6 m concentrationr ange (Figure 8). The characteristic SERS bands of 2 4 + + were obtained at ac oncentration as low as 5 10 À8 m,w hereas the Ramans cattering enhancementw as the highest for the 1 10 À6 m sample. Referring to the Raman bands assignment, the strongb and at 2215 cm À1 was assigned to the stretching of the CCt riple bonds, whilet he intense band at 1596 cm À1 was attributed to the stretching of the phenylm oieties. The mediumb and at 1354 cm À1 was assigned to the stretching modes of the central conjugatedp art of the molecule, including phenyl rings and triple bonds, whereas the moderate band at 1070 cm À1 wasa ssociated with the stretching of the bonds between the boron atom and aromatics ubstituents. Aside from the few 2 4 + + bands observed in the Ramans pectra,s ome additional bands were obtained in the SERS spectra, corresponding mainly to phenyl ring vibrations (Table S4). The bands at 1286 cm À1 and 1155 cm À1 which appeared at low 2 4 + + concentrations (1 10 À7 m and 5 10 À7 m) were attributed to the symmetric stretchings of -CF 3 and -SO 3 Figure 7. a) Raman spectrao f2 4 + + (c = 1 10 À4 m)inw ater (blue line) and Na-cacodylateb uffer,pH7.0 (red line). b) SERS spectraof2 4 + + (c = 1 10 À6 m), in the silver colloid not containingNa-cacodylate buffer (blue line) and containingNa-cacodylate buffer,pH7.0 (red line). l ex = 758 nm. The bandsl abelled with asterisks originate from the buffer. The spectraare displaced for visual clarity. groups,r espectively,o ft rifluoromethanesulfonate anions. [35] At low concentrationso f2 4 + + ,t he counterions came close to the enhancing silver surfacea nd, consequently,t heir vibrational modes were enhanced.
Considering the surface selection rules according to which polarizability changes perpendicular to the metal surfacec ontribute the most to scattered radiation, the prominent CC (2215 cm À1 )a nd phenyl (1596cm À1 )s tretching bands implied that, at ac oncentration of 1 10 À6 m,t he molecules adopted an optimal position with the triple bonds and phenyl rings oriented perpendicular to the silver surface (Scheme 3a).
Moreover,t he positive chargeo ne ach side of the molecule facilitated its positioningi nh ot spots between two nanoparticles. It can be assumed that adsorption of the 2 4 + + molecules was electrostatically driven by the positively charged trimethylamino groups attracted to the citrate anionso nt he silver nanoparticles, followed by direct interactions with the silver surface. Ab and observed in the low wavenumber region (216 cm À1 )was assigned to AgÀNstretching, indicating interaction between the nitrogen atom andt he silver surface ( Figure 7b). By decreasing the concentration, the intensity of the characteristic vibrational bands diminished, most likely due to first tilted and then in-plane positioning of the molecules on the enhancing surface( Scheme 3b).
In order to investigate the effect of the buffer on the SERS response, the spectrum of 2 4 + + (1 10 À6 m)i nN a-cacodylate buffer (pH 7.0) was acquired (Figure 7b). In general,t he spectrum was slightly more intense and the bands more defined, when comparedt ot he spectrum of 2 4 + + in water,t houghp ositively charged 2 4 + + molecules most likely acted as aggregating agents of the silver colloid. Salts in the buffer composition additionally induced aggregation of the silver nanoparticles resulting in stronger enhancement of the Ramans cattering. A strong band observed at 231 cm À1 was assigned to the stretching of the AgÀCl bond formed between the chloride ions from the buffer and the silver surface (Figure 7b).
The SERS bands were more intensef or the 2 4 + + /ct-DNA complex than for 2 4 + + alone. For example, at c(2 4 + + ) = 5 10 À8 m,t he intensity of the triple CCs tretching band at 2214 cm À1 for the complexes of r[2 4 + + ]/[ct-DNA] = 1a nd 0.2 was enhanced 1.4 and 1.3 times, respectively,r elative to that of the free 2 4 + + molecules. The increase in intensity obtained indicated interactions of 2 4 + + with the nucleic acid, owing to which the smallm olecules adopted am ore optimal orientation with respectt ot he enhancing silver surface and/or were placed closer to the silver nanoparticles (Scheme 3c). On the other hand, the excess of highly negatively charged DNA in the 2 4 + + /ct-DNAs ample of the r = 0.1 most likely caused lesse fficient adsorption of the complex on the silver surface, reducing the SERS intensity.
Furthermore,t he SERS response of 2 4 + + (c = 1 10 À6 m)u pon binding to BSA was studied for the 2 4 + + /BSA complexes prepared in r[2 4 + + ]/[BSA] molar ratios of 1, 0.2 and 0.1 (Figure 9c). Unlike the complexeso f2 4 + + with the nucleic acids, the SERS intensity significantly diminished regardless of the complex molar composition. The characteristicb and of the triple bond stretching at 2215 cm À1 almost completely vanished from the spectrum of the 2 4 + + /BSA complexo ft he molar ratio 0.1. Due to strongi nteractions of the small molecules with the protein, and likely deep insertion of the molecule within the BSA binding site, compound 2 4 + + was removed from the silver nanoparticles responsible for the SERS effect.
It is interesting to notet hat the characteristic SERS response of 2 4 + + was observed upon NIR excitation (785 nm) in the simplyp repared and widely used silver colloid,e ven at an anomolar concentration range. Thereby the distinctive band of the CCt riple bond at 2215 cm À1 ,w hich does not interferee ither with the bands of its own, or with the bands of the other species, in the measured system, allowed easy detection of the small molecules. The intense SERS response obtainedf or 2 4 + + interacting with DNA, and its loss upon binding with BSA, is characteristic of binding with the nucleic acid and the protein, respectively.O wing to the sensitivity obtaineda nd minimal spectrali nterference, the molecules studied could potentially be used as alkyne-coded SERS tags for live cell imaging. [2d] Scheme3.SERS experiments:Depictionoft he molecularo rientation of compound 2 4 + + with respect to the silver surfaceath igh concentrations of 2 4 + + (a), low concentrations of 2 4 + + (b), and in the complexw ith ds-DNA (c). The 2 4 + + /BSA complex (not shown) is detached from the Ag-surface and, thus, gives no SERS signal.

Preliminary biologicalscreening
The biological experiments aimed to verify the actual capability of the DNA/RNA/protein binder studied to penetrate the cells, to visualize its intracellular location and subcellular targets. Evaluation of the anti-proliferative effectw as conducted in order to identify further potentiala pplications, either as a cytotoxic lead compound towardt heranostic [36] applications (combining dual fluorescent/Raman monitoring with an antiproliferative action), or as an on-cytotoxic dye suitable for intracellularapplications or in biochemical studies.
To examine toxicity, i.e.,the effect of 2 4 + + on the proliferation of human cell lines,w eu sed the MTT test. The compound was tested on two human cell lines (HeLa and HEK 293). As an indicator of anti-proliferation activity we used the IC 50 value, which corresponds to the concentration of the compound that inhibits proliferation to 50 %c ompared to the control cells (proliferating without the compound in the medium). The results we obtained showedn egligible anti-proliferative activity of 2 4 + + even at the highest concentration used in the test (1 10 À4 m, results not shown).
We also checked the ability of the compound to cross the cellular membrane and penetrate the HeLa cells, by using the intrinsic fluorescenceof2 4 + + in fluorescent confocal microscopy experiments. Confocalm icroscopy also allows us to propose the possible intracellular localization of the compound. The compound 2 4 + + enteredc ells very efficiently within 2h ours of cell immersion in c(2 4 + + ) = 1 10 À6 m.T he observed fluorescence of the compound coincided with the area of the cell where the endoplasmic reticulum is located, and the grainy dispersion of the fluorescence signal indicates possible endosomal accumulation( Figure 10).
As titration experiments of 2 4 + + with DNA/RNA and BSA revealed strong quenching of the emission of 2 4 + + upon binding, the strong fluorescencei nt he cell suggests that emission from the compound is not significantly affected by its proposed intracellularl ocalization. This would be consistent with the hydrophobic endosomal environment, which is devoid of any DNA/RNA or albumin-like binding sites. This could spur intriguing future studies of 2 4 + + andi ts analogues in dual fluorescence/Raman-based SRS microscopy, [2d] whereby the intracellular location with quenched fluorescencea nd activeS RS signal could be easily differentiated from the intracellular location with strong fluorescence.
The cell nucleusisc ompletely void of any emission which, in combination with its negligible anti-proliferative activity, strongly suggestst hat 2 4 + + does not bind to or interferew ith genomic DNA or RNA processes, which are essential for cell viability.H owever,f or am ore accurate determination of its cellular localization, further, more detailed biological experiments are planned.
It can be safely assumed that the positivelyc harged analogue 2 4 + + retains structural features very similar to those of its neutrala nalogue 2N (see Figure 1);t hus, both compounds could be considered as rod-like dumbbell structures. Com-  pound 2 4 + + is also characterizedb yf our terminalp ositive charges. Only 2 4 + + was soluble in water and, therefore, further studied for biorelevant applicationsu sing, as ar eference, the correspondingmonomer 1 2 + + .
Both cationic compounds (2 4 + + , 1 2 + + )s howedr emarkably high affinity toward various types of DNA/RNA (logKs = 6-7.5 range) and, intriguingly,d icationic monomer 1 2 + + showedo nly an ordero fm agnitude lower affinity than tetracationic 2 4 + + .T his implies only am inor contribution of electrostatic interactions with the negatively charged DNA/RNA backbone and suggested binding within ds-DNA/RNA grooves, within which the highly hydrophobic linker and extensive interactions of aromatic units surrounding the borona toms strongly contribute to overall DNA/RNA affinity.
However,t etracationic 2 4 + + also strongly interacted with ss-RNA, which does not possess any groove as ab inding site. The CD results strongly supported ss-RNAc hain wrapping aroundt etracationic 2 4 + + as at hread around the spindle, which is av ery unusual mode of binding of ss-RNA with small molecules. The absence of interaction of ss-RNAw ith globularshaped structure of 1 2 + + additionally stressed the nature of the rod-like dumbbell structure of 2 4 + + being crucial for strong interaction with ss-RNA.
Another major designf eature of 2 4 + + was the ability to use the diyne-linker as aR aman-probe, complementing the 2 4 + + fluorescencer esponse. Thus, in aqueous solution 2 4 + + gave rise to severalr emarkably strong Ramanb ands in the 2220-1355 cm À1 range, allowing accuratem onitoring at as low as 10 micromolar concentration.E ven more interesting was the response of 2 4 + + in the SERS spectra, leading to ad etection limit below 10-nanomolar concentrations. Most intriguingly, addition of DNA actually increased the SERS signal intensity slightly,w hereas BSA completely quenched it. This specific response is very useful and complementary to that of the 2 4 + + fluorescencer esponse in which DNA/RNA and BSA cause similar emission quenching.
Finally,p reliminary biological activity screening showed that 2 4 + + entered human cells very efficiently while not interfering with cell viability up to 10 À4 m concentrations. Its bright fluorescencei sp roposed to originate from localization along the endoplasmic reticulum, possibly via intra-ribosomal or endosomal accumulation.
Our results strongly support furtherdevelopment of bis-triarylborane Raman/fluorescence chromophores as dual probes for simultaneous confocal fluorescence microscopy and SRS microscopy of cell lines, whereby careful choice of al inker can finely tune DNA-protein selectivity (notet he difference between 2 4 + + diyne vs. 3 4 + + bithiophene) and consequently the intracellulara ccumulation of ap robe. TheR aman-response of 2 4 + + -analogues in SRS microscopy could, thus, efficiently complement the fluorescencer esponse when the studied system is turbid/non-suitable for fluorescenceimaging (e.g. cell-organoid agglomerates), some other fluorescencep robe is used simultaneously,o ro ne of the cellular targets quenchest he fluorescence emission. Moreover,t he unique bindingm ode of ss-RNAs with very high affinity makesb is-triarylborane tetracations very promising ss-RNA delivery systems, particularly as some of them have already shown very efficient cellular uptake and negligible toxicity. [17p, 19, 26]