# PNA aptamer-based bioreceptors for cardiac biomarker (cTnI) detection: Insight into the structural and stability-related aspects

Francesco Basini, Abdellah Hambli, Subhankar Sahu, Rupali Bagale, Manova

Santhosh Yesupatham, Christophe Ritzenthaler, David Montaigne, Eloise

Woitrain, Henri Happy, Rabah Boukherroub, et al.

► To cite this version:

Francesco Basini, Abdellah Hambli, Subhankar Sahu, Rupali Bagale, Manova Santhosh Yesupatham, et al. PNA aptamer-based bioreceptors for cardiac biomarker (cTnI) detection: Insight into the structural and stability-related aspects. Nano Today, 2026, 67, 10.1016/j.nantod.2025.102957. hal-05425091

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# PNA aptamer-based bioreceptors for cardiac biomarker (cTnI) detection: Insight into the structural and stability-related aspects

Francesco Basini<sup>a,1</sup>, Abdellah Hambli<sup>b,c,1</sup>, Subhankar Sahu<sup>b,d,*</sup>, Rupali Bagale<sup>b,c</sup>, Manova Santhosh Yesupatham<sup>c</sup>, Christophe Ritzenthaler<sup>d</sup>, David Montaigne<sup>e</sup>, Eloise Woitrain<sup>e</sup>, Henri Happy<sup>b,2</sup>, Rabah Boukherroub<sup>b</sup>, Wolfgang Knoll<sup>c</sup>, Sabine Szunerits<sup>b,c,*ID</sup>, Roberto Corradini<sup>a,**</sup>

## ARTICLE INFO

**Keywords:**

Aptamer

Biosensing

Peptide nucleic acids (PNAs)

Cardiac biomarker

Molecular dynamics simulations

## ABSTRACT

Aptamers, nucleic acid molecules that fold into specific three-dimensional structures, have been extensively used in the biosensing field to accomplish sensitive and specific monitoring of a wide range of biomarkers. Peptide nucleic acid (PNA), in this context, can be considered as a potential next-generation scaffold for aptamer synthesis and biomarker sensing, owing to its high stability in comparison to DNA counterparts. In this work, we investigated the performance of a series of PNA aptamers for monitoring of a prominent cardiac biomarker, cardiac troponin I (cTnI), using surface plasmon resonance (SPR) and showed that PNA sequences shorter than those previously reported for DNA can exhibit picomolar affinity, provided the essential structural features are preserved. Two different immobilization strategies (covalent and non-covalent) are validated in parallel for PNAs. The stability of sensor response in the presence of endonucleases such as DNase I was investigated further, as their occurrence in blood, plasma, and serum hydrolyses phosphodiester bonds and could be a limiting factor for point-of-care (PoC) application of DNA aptamers. Owing to their unnatural backbone, PNAs exhibited higher stability against DNase I in comparison to their DNA aptamer counterpart. Additionally, molecular dynamics (MD) simulations of DNA and PNA aptamers revealed similarities in their secondary structures, as well as distinctions in their propensity to adopt compact conformations. Overall, our findings not only provided a comprehensive framework for PNA design, surface functionalization, and cTnI biomarker detection using PNA-based bio-recognition scaffolds but also substantiated the biostability of PNAs, suggesting their high relevance for future PoC diagnostic applications.

## Introduction

Accurate and timely detection of blood-derived biomarkers through diagnostic devices has become a highly valued early-warning approach for disease risk evaluation and monitoring therapy progress [1]. Protein biomarkers, in particular, serve as pivotal indicators for tracking inflammation, immune response, and progression of diseases like

cancer, cardiovascular syndromes, as well as neurological disorders [2, 3]. The identification of diagnostic biomarkers holds a key potential for personalized medicine, leading to enhanced clinical outcomes, and is closely linked to the development of sensitive and selective diagnostic approaches [4]. Personalized medicine is an unmet clinical need in cardiovascular diseases (CVDs) such as heart failure (HF), which is caused by a variety of cardiac and extracardiac pathophysiological

<footnote><sup>a</sup> University of Parma Department of Chemistry, Life Sciences and Environmental Sustainability, Parma 43124, Italy</footnote>

<footnote><sup>b</sup> Univ. Lille, CNRS, Univ. Polytechnique Hauts-de-France, UMR 8520 - IEMN, Lille F-59000, France</footnote>

<footnote><sup>c</sup> Laboratory for Life Sciences and Technology (LiST), Faculty of Medicine and Dentistry, Danube Private University, Krems 3500, Austria</footnote>

<footnote><sup>d</sup> Institut de Biologie Moléculaire des Plantes, UPR2357 du Centre National de la Recherche Scientifique, Université de Strasbourg, Strasbourg F-67084, France</footnote>

<footnote><sup>e</sup> Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011- EGID, Lille F-59000, France</footnote>

<footnote>* Corresponding authors at: Univ. Lille, CNRS, Univ. Polytechnique Hauts-de-France, UMR 8520 - IEMN, Lille F-59000, France.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: subhankar.sahu@univ-lille.fr (S. Sahu), sabine.szunerits@univ-lille.fr (S. Szunerits), roberto.corradini@unipr.it (R. Corradini).</footnote>

<footnote n="1">¹ contributed equally</footnote>

<footnote n="2">² In memory of Henri Happy, whose spirit continues to inspire.</footnote>
mechanisms, leading to diverse clinical phenotypes and outcomes. Moreover, in cardiovascular emergencies, such as acute myocardial infarction (AMI) and acute HF, timely diagnostics can be difficult because the clinical symptoms are often nonspecific and may have limited diagnostic value. Currently, protein biomarkers used to predict the prognosis of chronic HF and patients facing acute cardiac conditions are either released from the heart or other cells as a systemic response to HF with natriuretic peptides (e.g. brain-type natriuretic peptide (BNP) and N-terminal prohormone of BNP) as well as cardiac troponin (cTnI) measurements being part of the guidelines of HF diagnosis of the European Society of Cardiology [5]. Most commonly, these proteins are detected in patient blood in a laboratory setting using antigen-based enzyme-linked immunosorbent assay (ELISA) concepts, which are, however, rather lengthy and often not adapted for blood dosing in emergencies. Significant efforts have been made in recent years to develop fast-sensing technologies. However, many current biosensors lack the sensitivity and selectivity required for cardiac targets, where detection limits in the low pg mL⁻¹ range are necessary, levels well below what most diagnostic sensors can achieve [6,7].

One way to address this challenge is to consider the benefits of aptamers over antibodies as reliable molecular recognition elements. Aptamers offer low manufacturing costs, limited batch-to-batch variation, a reversible folding feature, long shelf time (several years), and increased thermal and chemical stability compared to monoclonal antibodies, while maintaining high target affinity and specificity [8,9]. Although DNA aptamers have many merits, their susceptibility to nuclease degradation is a known limitation [10,11]. The problem of nuclease-induced degradation is particularly complex during the early stages of acute myocardial infarction since it was shown that DNase I levels can significantly increase during these phases [12], thus leading to increased jeopardy for DNA-based sensors. Some similar concerns were recently raised during stem cell transplantation [13]. While some studies reported on retarded degradation of surface-bound ssDNA by DNase I [14,15], most investigations underlined the instability of surface-bound DNA to DNase I [16,17]. Therefore, developing alternatives to DNA-based aptamers is expected to significantly improve real-time measurements of cTnI in biological fluids. In this respect, modification of the aptamer chains and/or conjugation with functional molecules is required.

Over the years, a series of techniques has been established to protect DNA probes, including aptamers, from nuclease degradation based on modification of the 3' and 5' ends of the oligonucleotide, the sugar ring, and/or the phosphodiester backbone [9,18]. The majority of the aptamers clinically applied are therefore modified by capping the 3'-end with inverted thymidine [19]. Modifications of ribose (e.g. 2'-F, 2'-NH₂, 2'-OMe substitutions) or replacement of ribonucleotide analogues with locked nucleic acid (LNA, linking 2'-O and 4'-C of the ribose with a methylene bond) have been considered in addition [20]. The application of spiegelmers to prevent aptamers from nuclease degradation has also been put forward as an effective strategy [21]. Zhao *et al.* addressed this issue for DNA-based tension sensors using peptide nucleic acid (PNA) binder rather than DNA [11]. It was indeed confirmed in this study that a PNA/DNA hybrid is DNase I-resistant and remains stable in DNase-expressing cells. While this and other works underlined that capture probes based on PNA backbones are promising alternatives to standard oligonucleotides for hybridization studies [22–24], the use of PNA aptamers for protein biomarker sensing has only recently been proposed [25].

There have been some recent efforts to understand the cTnI-DNA aptamer complex; however, not much is known about PNA scaffolds and structural aspects related to cTnI binding. The interaction between the hairpin structure of a series of DNA aptamers (Tro-1 to Tro-6) and cTnI was previously investigated by Ropil et al. [26] using molecular docking and molecular dynamics (MD) simulations, revealing specific sequence clusters in the aptamer that play a crucial role in cTnI binding. In a related study, Mokhtari et al. [27] demonstrated that several

aptamer bases form hydrogen bonds with cTnI residues, while others participate in hydrophobic and π-π stacking interactions across a wide range of binding sites. These interactions were shown to significantly enhance the stability of the DNA–cTnI complex. Nevertheless, extrapolating these findings to PNA aptamers is not straightforward, due to structural and chemical differences, highlighting the need for direct experimental evaluation of PNA–cTnI interactions. In a separate approach, Qin et al. [28] investigated the use of truncated DNA aptamers to improve the sensitivity of cTnI detection by shortening the original Apt-40 sequence to 38, 36, and 34 nucleotides. Although these results demonstrated enhanced sensor sensitivity, they lack insights into quantitative binding affinity or characterization of specific interaction sites between the aptamer and cTnI. At this juncture, further studies are required, especially for the PNA-based scaffolds, to better understand the structural context of aptamers and resulting cTnI recognition sensitivity.

In this work, we build upon our recent research on DNA and PNA aptamers interfaced with electrolyte-gated graphene-based field-effect transistors (gFET) for cardiac troponin I (cTnI) sensing [25,29,30] and further advance the work by exploring the structural and stability-related aspects of PNAs. We designed a series of PNA aptamers of different lengths and terminal groups, screened their binding kinetics profiles for the cTnI biomarker, which allowed us to decode the structural aspects of the PNA scaffold and identify a motif facilitating the cTnI recognition (Fig. 1). The complete PNA aptamers showed picomolar order binding affinity for cTnI. The PNA stability against endonucleases such as DNase I was evaluated in addition using surface plasmon resonance-based studies, which established the PNAs to be more robust than the DNA aptamers and positioned them as highly effective bio-receptors for real sample analysis.

## Results and discussion

### Conformational dynamics studies of PNA and DNA

The conventional iterative selection method, such as the Systematic Evolution of Ligands by EXponential Enrichment (SELEX) process, is fundamentally unsuitable for PNA aptamer selection due to the distinct backbone chemistry of PNA compared to DNA. We have, however, shown previously that the sequence of a known DNA aptamer can be used in some cases for the construction of PNA aptamer analogues, while maintaining strong binding capacity to the target analyte [25]. This approach cannot be generally used, especially when electrostatic interactions are crucial for protein binding and considering that PNA does not feature the same negative charge as DNA. Therefore, in order to design new PNA aptamers, it is important to understand the underlying conformational dynamics and the similarity in the binding sites, which can be important for deciphering interactions specific to target recognition.

In our work, all the aptamers display a simple nomenclature according to their length. The Apt-40 [28] is referred to as DNA-40, and the analogous PNA version containing identical bases is referred to as PNA-40. The characterization details of all the synthesized PNAs investigated in this work are represented in Figures S1-S19. In order to be able to rationalize the interactions between the different segments of aptamer PNA-40 and the cTnI protein, we first tried to understand the structural characteristics of this system and compared it to its DNA counterpart. Unfortunately, no experimental structures are available till date for the DNA-40, PNA-40, or their complexes with cTnI. Moreover, structural studies on single-stranded PNA are scarce in the literature. Therefore, first, we used MD simulations to predict conformational dynamics and compared the behavior of the preassembled stem-loop structure for both PNA-40 and DNA-40 scaffolds.

The structures of aptamers DNA-40 and PNA-40 were prepared as described in materials and methods and simulated for 1 µs using two different force fields (FFs) for comparison [31]. The four MD trajectories
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Schematic illustration of the biosensing of cTnl biomarker by a PNA aptamer functionalized portable SPR platform. A series of PNA aptamers and their structural aspects are studied for cTnl binding. The PNA aptamers are more stable against the common nucleases like DNase I than the conventional DNA aptamers, making them more suitable for PoC sensing in blood samples. The visuals provided are not drawn to scale and are intended for representation only.</figcaption></figure>

(DNA/Amber, DNA/CHARMM, PNA/Amber, PNA/CHARMM) were analyzed using VMD [32] and the root-mean square deviation (RMSD) with respect to the starting structure (Figure S20) as well as the root mean square fluctuation (RMSF) over the 40 residues of each oligonucleotide chain (Fig. 2A-B) were reported to assess global stability and local flexibility. The RMSD values indicate that the initial stem-loop structure predicted for both the DNA-40 and PNA-40 is stable within the 1 µs simulation trajectory, with minor variations between the two FFs and with respect to the starting one, indicating the stability of the stem-loop motif hypothesized for DNA. The dynamics were stable after ~200 ns, with RMSD fluctuating around 10 Å (Figure S20).

To identify the most flexible part of the aptamers, we looked into the RMSF of each residue among the four trajectories. RMSF analysis (Fig. 2A-B) reveals significant mobility in the loop region, in the bulge, and in the long-end tail. For PNA-40, motion is primarily localized within the loop segment, from residue 15–25, at the two bulge residues in the middle of the stem, residues 8 and 9, and at the unpaired terminal residues. The RMSF profile highlights the stem region as the most stable portion of the molecule, and the PNA scaffold has overall less fluctuation. The structural snapshots extracted from the MD (Fig. 2C-F) reveal that DNA-40 tends to adopt a more rigid and stretched conformation

(Fig. 2C-D), whereas PNA-40 is more compact and flexible. This difference can be attributed to the absence of electrostatic repulsion in the neutral PNA backbone, which lacks the charged phosphate groups present in DNA. As a result, attractive base–base interactions in PNA are less counterbalanced, and its neutral, more apolar backbone promotes stronger hydrophobic collapse (Fig. 2E-F).

The simulation of DNA/CHARMM shows a stem–loop structure that is overall stable, although some stem bases tend to break Watson–Crick pairing, leading to π–π stacking interactions (Fig. 2D). In this trajectory, the conformation remains rather rigid and elongated, and the RMSD plot exhibits larger fluctuations, around 13 Å (Figure S20B). The RMSF profile does not clearly highlight markedly more stable regions, except for the terminal part of the stem between bases 25 and 32 (Fig. 2B). The PNA/CHARMM structure is more compact (Fig. 2 F) with a stable stem, and motion is localized at the unpaired terminal residues as depicted in the RMSF plot (Fig. 2B). However, the relatively high RMSD in this case, around 16 Å, is mainly due to movements of the segment between residues 32 and 40, which often form π–π stacking interactions with the loop bases (Figure S20B).

These simulations demonstrate that the stem–loop architecture hypothesized for PNA-40, by analogy with the structure predicted for DNA-

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** (A) Root-mean square fluctuation (RMSF) per residue for the Amber trajectories: DNA aptamer (blue, line and markers), PNA aptamer (red, line and markers). (B) RMSF per residue for the CHARMM trajectories: DNA aptamer (light blue, line and markers), PNA aptamer (yellow, line and markers). (C-F): representative snapshots extracted at 500 ns from the 1000 ns MD simulations; backbone ribbons are colored as in the RMSD/RMSF plots. (C) DNA/Amber, (D) DNA/CHARMM; (E) PNA/Amber, and (F) PNA/CHARMM. Atoms and bonds are displayed in licorice. PNA and DNA structures were visualized and rendered with UCSF Chimera and UCSF ChimeraX, respectively [33,34]. Residues colored in green within structures (E) and (F) represent the PNA sequence defined by PNA-20.</figcaption></figure>
40, remains stable over the simulation timescale. It is therefore plausible that a stem-loop-folded PNA-40 interacts with the target cTnI with similar affinity as DNA-40. The insight from these 3D structures allowed us to further rationalize the structure-activity dependence of the designed PNA library, also considering the spatial disposition of the interacting sites (as discussed in the following Section 2.3). Furthermore, to the best of our knowledge, this is the first atomistic simulation of a single-stranded PNA (ssPNA) performed using the optimized backbone torsion parameters for PNA of Jasiński et al. [31]. These parameters were reported to preserve structural stability over microsecond-scale MD and to reproduce quantum-mechanical (QM) torsional energy surfaces for the PNA backbone. Moreover, this is the first comparative analysis of the conformational dynamics of ssPNA and ssDNA pre-assembled into a defined secondary structure.

## Library of PNA aptamers

In order to gain a detailed insight into the cTnI recognition with PNA bioreceptors, a series of PNA aptamers of varying lengths was designed to investigate their interaction with cTnI by SPR (Fig. 3). The PNA-40 is the full-length reference aptamer containing a hairpin (stem-loop) structure with complementary base pairs forming the central stem region and a single-stranded tail (Fig. 3A). Shorter variants (PNA-38 to PNA-27A) exhibited truncated structures characterized by progressively reduced tails. The PNA-27B, PNA-20, and PNA-15 represent the second part of the aptamer, starting at the 14th and 17th base within the loop region, respectively (Fig. 3B-C), while PNA-17 corresponds to the first part of PNA-40, complementary to the PNA-20. Additionally, we

included PNA-20r and PNA-14r as negative controls, which are essentially fully disordered scrambled sequences.

## Binding affinity evaluation of the PNA aptamer library

The binding affinities of the PNA aptamers for cTnI were quantitatively evaluated by surface plasmon resonance (SPR) (Fig. 4). For these measurements, all the PNAs were designed to contain an N-terminal azide group (N₃-), which is exploited for the surface immobilization onto the SPR prism (gold surface), as shown in Fig. 4A.

A copper-catalyzed azide-alkyne cycloaddition (CuAAC) [35] reaction was used to “click” the azide terminus of the PNAs with the surface-exposed 4-pentynoic acid groups. Binding affinity measurements revealed that PNA-40 exhibited the highest affinity for cTnI with a K<sub>D</sub> = 1.3 pM (Fig. 4B). The PNA-38, 37, and 35, having a shorter front tail part, exhibited similar affinities with K<sub>D</sub> values = 1.7, 1.7, and 2.5 pM, respectively. The sensorgram profile of each of the N₃-PNA aptamers is depicted in Figure S21. These aptamers share a conserved cluster of 24 contiguous nucleobases near the tail region (CTTT-CTCA-TGCG-CTGC-CCCT-CTTA), indicative of a structural or sequence-specific core critical for target recognition. Progressive truncations of this downstream region (PNA-29, PNA-27A, PNA-27B, and PNA-20) resulted in a gradual decrease in binding affinity, with K<sub>D</sub> values increasing from 3.6 to 8.4 pM. PNA-20 offers the optimal balance between length and affinity: it maintains picomolar target affinity while using only half the number of nucleobases compared to the DNA-40 original aptamer, reducing both synthesis cost and complexity. Further deletions removing terminal motifs within this core (PNA-15)

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Structure of PNAs explored in this work. A series of different PNA aptamers and their predicted secondary structures were investigated towards cTnI biomarker sensing. (A) The PNA-40 is regarded as the complete aptamer exhibiting a stable hairpin structure with a loop and an extended tail. (B-C) Predicted secondary structures and sequences of all the other PNAs are provided along with their names. All the aptamers carry an N-terminal azide (N₃) function, and a C-terminal glycine-amine denoted as R (Gly-NH₂). The PNA-14r includes a spacer sequence at the beginning. Predicted secondary structures of all the PNAs were obtained using RNAfold [48].</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 4. Surface functionalization approach for immobilization of N₃-PNA aptamers and binding affinity measurements. (A) Step1: Formation of a self-assembled monolayer upon interaction of the thiol function of L-cysteine (L-Cys) with gold. Step 2: Amide bond formation between the NH₂ groups of L-Cys and the COOH groups of 4-pentynoic acid (4-PA) after activation of 4-PA with EDC/NHS. Step 3: copper-catalyzed azide-alkyne cycloaddition reaction between N₃-PNA and alkyne group (≡C-H) on gold surface. Step 4: The SPR platform is ready for sensing of cTnI by the PNA aptamer surface. Comparison of (B) dissociation constants (K_D), (C) k_on and (D) k_off rates for interaction between the azide (N₃)-PNA aptamers with cTnI as determined by SPR. In SI, these PNA aptamers are identified as N₃-PNA-"number". For enhanced clarity, the values corresponding to each case are displayed above the respective error bars within the plot.</figcaption></figure>

led to significantly weaker binding, K_D = 37.6 pM (Fig. 4B). To validate the importance of this part of the aptamer, we also tested the other half of the 40-mer sequence with respect to PNA-20 (PNA-17), and it showed no detectable binding. As negative controls, we also tested a sequence unrelated to the parent PNA (PNA-14r) and detected no affinity. In addition, to confirm that PNA-20's affinity is sequence-dependent rather than due to base composition, we prepared a scrambled variant with identical base composition in randomized order (PNA-20r), which likewise showed no detectable affinity.

Collectively, these findings suggest that a 20-nucleotide cluster near the tail region (corresponding to PNA-20) might be the critical binding core indispensable for high-affinity recognition of cTnI. The sequence closer to the loop likely contributes to structural stabilization but is insufficient to confer binding without this core sequence. While a stable hairpin structure in longer aptamers may enhance binding affinity by stabilizing the aptamer's conformation, it is not strictly necessary for effective target recognition. Shorter variants containing the critical cluster sequence still demonstrate measurable binding, indicating that the essential binding interactions can occur without the full hairpin architecture. This suggests that the affinity depends more on the exposure and accessibility of key binding motifs rather than solely on the presence of a stable stem-loop structure. Further structural and biochemical studies will be essential to elucidate the precise conformational features that govern this specific interaction, and a full understanding would require structural data on the various PNA-cTnI complexes. However, it is worth noticing that the residues contained in PNA-20 define a specific region in the MD calculated 3D structures

(Fig. 2E-F), indicating the side of the PNA aptamer involved in the interaction with the protein. This spatial localization helps identify the binding interface and provides insight into how PNA-20 likely engages its target at the molecular level. The full-length PNA-40 has a secondary structure that is able to maintain these residues in a preorganized pattern, thus giving rise to maximum affinity. However, the flexibility of the PNA backbone could allow the shorter segments used in the present study to adapt to a suitable conformation able to maintain the same PNA-protein interactions with an 'induced fit'-kind model and thus preserve a very high affinity for the target.

The selectivity of the PNA aptamer was, in addition, studied (Fig. 5A, Figure S22). It could be shown that other cardiac biomarkers, such as Cardiac Troponin T (cTnT) and B-Type Natriuretic Peptide 32 (BNP-32), revealed no interaction with the PNA modified SPR, underlining good selectivity. Important to acknowledge is that the PNA aptamers, similar to other PNA-based biorecognition elements, may exhibit low-level interactions with non-specific analytes, as has been reported for other PNA bioreceptors [36,37]; nevertheless, within our sensor design, the non-specific signal from the other related biomarkers was negligible.

Furthermore, the sensitivity of SPR chips modified with full PNA-40 compared to truncated PNA-20 for cTnI was evaluated. Standard calibration curves extracted from the steady-state response of the SPR signal reveal that the RU response increases proportionally with cTnI amount and saturates around 5 ng mL⁻¹ of protein concentration (Fig. 5B-C). A limit of detection (LOD) for cTnI was extracted to be 11 pg mL⁻¹ (PNA-40) and 40 pg mL⁻¹ (PNA-20) respectively. The higher LOD in the case of PNA-20 as bioreceptor is in line with the higher binding affinity of the
<figure><img src="image_6.png" /><figcaption>Fig. 5. Selectivity and Sensitivity considerations. (A) Selectivity profile of the PNA aptamer when tested with various other cardiac biomarkers, including Cardiac Troponin T (cTnT) and B-Type Natriuretic Peptide 32 (BNP-32). The cTnI sensing signal to SPR chips modified with (B) PNA-40 and (C) PNA-20 aptamers extracted from SPR experiments at steady state response.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 6. Affinity measurements of cTnI with pyrene-PNA (py-PNA) aptamers. (A) Illustration of the surface chemistry immobilization approach of the py-PNA aptamers over graphene-coated gold prisms. The pyrene group at the N-terminal of the PNA is used for the immobilization on the graphene surface by π-π stacking interactions. The py-PNA-coated surface is then used for cTnI sensing by SPR. Comparison of (B) dissociation constants ($K_D$), (C) $k_{on}$, and (D) $k_{off}$ rates for interaction between the py-PNA aptamers with cTnI in solution, as determined by SPR. Different concentrations of cTnI (5–50 pM) in 1 × PBS-TMg (1 × PBS, 1 mM MgCl$_2$, and 0.05 % v/v Tween-20) running buffer are tested for each aptamer. For enhanced clarity, the values corresponding to each case are displayed above the respective error bars in the plot.</figcaption></figure>
PNA-20 scaffold to cTnI. (Fig. 4).

Following the evaluation of N₃-PNA modified aptamers, we investi-
gated pyrene-modified counterparts of some of the strongest PNA
aptamer binders to determine whether changing the surface anchoring
chemistry would affect the binding performance. This is relevant,
especially considering pyrene-modified aptamers are attractive from the
perspective of their implementation within graphene field effect tran-
sistor (gFET)-based sensing actuators [38]. While the N₃-PNA constructs
were covalently attached (Fig. 4A), the pyrene-modified aptamers
(py-PNA) interacted with graphene-coated SPR chips via π-π stacking
(Fig. 6A). When tested for cTnI detection, their binding affinities
remained in the lower picomolar range and showed minimal variation
(Fig. 6B). The py-PNA-40, 38, and 37 exhibited binding affinities of 1.5,
2.2, and 1.7 pM, respectively, comparable to their N₃-PNA counterparts
(Figure S23). Such robustness indicates that PNA-based sensors can
maintain consistent binding performance regardless of the surface
immobilization chemistry, positioning PNAs as versatile receptors
compatible with various sensing platforms.

Stability of the PNA aptamers against nucleases degradation

DNase I is one of the most common non-specific endonucleases in the
human serum, commonly used for DNA footprinting and non-specific
cleavage of duplex DNA. It displays a modest sequence- and structure-
dependent cleavage bias (for example, enhanced cleavage near certain
pyrimidine-purine bases), but it does not recognize strict primary-
sequence motifs [39,40]. DNase I is consequently considered more
effective at disrupting DNA strands than other nucleases such as
Exonuclease I (Exo I), EcoRV restriction endonuclease, micrococcal
nuclease (MNase), and S1 nuclease [41]. Earlier studies reported DNase
I blood concentration in healthy patients of about 3.2 ± 1.4 ng mL⁻¹
[42], while during early stages of acute myocardial infarction, DNase I
concentration can increase several-fold [12], with a time-dependent

decline within 12 h and returning to basal levels within 24 h. Building
on these previous findings regarding DNase I concentration, activity,
and clinical relevance, we next sought to evaluate its effect on sensing
performances. To probe the impact of the presence of DNase I activity on
the affinity of PNA and DNA aptamers towards cTnI, aptamer-modified
SPR prisms were exposed to a series of different DNase I concentrations
(5–20 ng mL⁻¹) [12,42] for 2 h, before the cTnI binding response was
measured. The DNA aptamer showed a high picomolar affinity for cTnI
(Figure S24A), as was also observed in our previous work [25], and the
interface exhibited a progressive loss in SPR signal with an increase in
DNase I treatment, with a near complete depletion of signal at a DNase I
concentration of 20 ng mL⁻¹ (Fig. 7A, S24B-D). Since DNase treatment
can alter the number and/or quality of functional surface receptors,
kinetic assumptions remain no longer reliable, and instead, we analyzed
the fractional RU (RU<sub>fractional</sub>) for 50 pM cTnI when the signal is satu-
rated (steady state, at 580 s). The signal loss was directly reflected in the
decreasing RU response (Figure S24E). From this, we obtained an
IC<sub>50</sub>-like DNase concentration of ~10 ng mL<sup>-1</sup> for loss of binding ac-
tivity, and we can infer that the DNase treatment affects the binding
capacity of the DNA scaffolds.

In contrast, the PNA aptamer retained more than 95 % of its SPR
signal even at the highest DNase I concentration tested, inferring no
significant degradation (Fig. 7B). Correspondingly, its binding affinity
remained remarkably stable, with K<sub>D</sub> values changing from 1.7 pM
(0 ng mL<sup>-1</sup>) to 2.9 pM (10 ng mL<sup>-1</sup>) (Fig. 7 C, S25). The minor increase
observed in the presence of DNase I, might be related to the presence of
weak nonspecific interference of the enzyme with the sensing interface.
Comprehensively, these results clearly demonstrate that PNA aptamers
preserve both signal output and target-binding ability under nuclease
treatment *in vitro*, whereas DNA aptamers are irreversibly degraded.
This also corroborates early studies by Demidov et al., who demon-
strated that due to the polyamide backbone, PNAs are highly resistant to
digestion after incubation with S1 nuclease and DNase I, and their

<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Influence of DNase I activity on stability of the DNA and PNA aptamers: (A) The RU response profile of the DNA aptamers reveals a significant decrease for cTnI binding when treated with DNase. (B-C) The PNA aptamers are stable against the nuclease activity and exhibit almost consistent K<sub>D</sub> values in the pM range. For enhanced clarity, the values corresponding to each case are displayed above the respective error bars in the plot.</figcaption></figure>
integrity is unaffected [43]. This, combined with our earlier studies on the stability of the PNAs across a large range of pH [25], establishes PNA aptamers as attractive bioreceptors in real environments and positions them as highly promising future scaffolds for PoC monitoring.

## Conclusion

Despite the rapid progress in healthcare biosensors, ranging from virus diagnostics [44,45] to wearable technologies [46], optimizing this evolving technology for real samples (blood, serum, or plasma) remains critically important. In this research, we explore the structural aspects of a new series of PNA aptamers targeted towards cardiovascular biomarker (cTnI) with pM order binding affinity and demonstrate their stability-related features. The PNA bioreceptors can sustain their binding affinity even when treated with DNase I, while the DNA versions lose the binding capability rapidly. This remarkable stability likely arises from the ‘peptide-backbone’-like structure of PNA, which resists DNase degradation while maintaining pM-order affinity for cTnI. This combination is crucial for long-term monitoring and especially important during the early phases of myocardial infarction. The PNA-40, 38, and 37 are highly sensitive for cTnI, with K<sub>D</sub> values being respectively 1.2, 1.7, and 1.7 pM when measured through a portable SPR platform. Furthermore, analysis of PNAs of varying lengths reveals that the nucleobase sequence within the 3’end part of the aptamer, in particular between bases 18 and 37 (PNA-20), rather than the hairpin structure, could be the decisive factor governing the interaction with the protein. In parallel, molecular modeling and dynamics suggest that the PNA-40 aptamer adopts the same stem-loop structure as also predicted for the DNA counterpart, while attaining a more flexible and compact conformation. Also, the non-covalent attachment of PNAs on graphene-modified surfaces does not compromise the cTnI sensing, showing their adaptability for graphene-based PoC actuators. Overall, this work highlights the potential and versatility of PNA aptamers as an emerging real-time CVD biomarker surveillance tool.

## Materials and methods

### Materials

The Fmoc-protected PNA monomers were purchased from PolyOrg, Inc. (Leominster, MA, USA). O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) was purchased from Fluo-rochem Ltd (Derbyshire, United Kingdom). N,N-diisopropylethylamine (DIPEA) was procured from Alfa Aesar (Kandel, Germany). Piperidine, GPR RECTAPUR, was obtained from VWR International srl (Milano, Italy). N,N'-diisopropylcarbodiimide (DIC), and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (DhBtOH) were purchased from TCI chemicals. Dichloromethane (DCM), N,N-dimethylformamide (DMF), and acetonitrile were procured from Carlo Erba Reagents (Milano, Italy). Rinkamide-ChemMatrix resin (35–100 mesh particle size) was purchased from ChemMatrix (PCAS BioMatrix Inc., Saint-Jean-sur-Richelieu, QC, Canada). Biotin, Boc-Gly-OH, trifluoroacetic acid (TFA), formic acid (FA), trifluoromethanesulfonic acid (TFMSA), azidoacetic acid, N-hydroxysuccinimide (NHS), pyridine, Fmoc-Gly-OH, thioanisole, m-cresol, acetic anhydride (Ac<sub>2</sub>O), diethyl ether, phosphate buffer tablets (PBS, 0.1 M, Cat. P4417), N-butyl hexafluorophosphate, tetrabutylammonium fluoride (TBAF), copper (II) sulfate (CuSO<sub>4</sub>), L-ascorbic acid, ethylenediaminetetraacetic acid (EDTA), 1-pyrenylbutyric acid, L-cysteine, sodium ascorbate, copper (II) sulfate pentahydrate, bathophenanthroline disulfonic acid disodium salt, and methoxypolyethylene glycol azide PEG (mPEG-N<sub>3</sub>, average M<sub>n</sub>=2000) were obtained from Sigma-Aldrich and used as-received. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from Alfa Aesar.

5'-azide modified cTnI DNA aptamer: 5'-N<sub>3</sub>-TTT-TTT-CGT GCA GTA CGC CAA CCT TTC TCA TGC GCT GCC CCT CTT A-3' was purchased

from IDT Technologies (USA). Recombinant human cardiac troponin I protein (M<sub>w</sub> = 24 kDa), recombinant human cardiac troponin T (M<sub>w</sub> = 36.4 kDa) and human DNase I ELISA Kit were obtained from Abcam (Cambridge, UK). BNP-32 peptide (M<sub>w</sub> = 3.4 kDa) was procured from BACHEM AG (Switzerland).

### PNA aptamers synthesis

**N<sub>3</sub>-cTnI PNA aptamer** was previously synthesized using Boc-chemistry [25]. In this paper, a new batch of PNA was produced using Fmoc-chemistry. This and all other azide-bearing PNA aptamers were synthesized using solid-phase protocols described previously [47]. The terminal azido group was introduced using coupling with 2-azidoacetic acid to the last PNA monomers, with long coupling times to ensure complete derivatization. Detailed descriptions of the synthesis and characterization of products are reported in the SI.

**Pyrene-PNA aptamers synthesis.** Terminal pyrene functionality was added after the deprotection of the last PNA monomer, via coupling 1-pyrenylbutyric acid. The coupling solution was prepared by dissolving in dry DMF, 1-pyrenebutyric acid (5 eq., 0.5 M), HBTU (4.9 eq. 0.49 M), and DIPEA (10 eq. 1 M). After 2 min of activation, the solution was added to the resin, and the coupling reaction proceeded for 4 h. The identity of the pyPNA-aptamer (40 bp) was assessed by MS analysis after purification by HPLC (see SI for purification protocols and product characterization). A stock solution of the probe in double-distilled water was prepared; the concentration of the probe was calculated from UV absorption at 260 nm. More details are reported in the SI. For modelling of 2D structures of the PNA aptamers, a similar approach of Ropil et al. [26] was followed. The structures were predicted by RNAfold [48] webserver, and the visualizations were carried out by Forna webserver [49].

### Surface modification of SPR Chips

#### “Click” chemistry for N<sub>3</sub>-PNA aptamers immobilization

Surface modification of the gold-coated prims for the N<sub>3</sub>-PNA aptamer immobilization was carried out by our earlier optimized protocol [25]. Briefly, first, the gold (Au) coated SPR chips were functionalized with L-cysteine (25 mM) outside the instrument for 30 min, washed, and air-dried. The chip was then coated with 4-pentynoic acid (4-PA), serving as an anchor for further aptamer immobilization. For this, the 4-PA was first activated with EDC/NHS (25 mM each in 1 × PBS) by mixing (30 mins) in a Hula rotary mixer, and then the solution was drop-casted over the L-Cys/Au prism and incubated for 2 h (at RT). Various PNA aptamers used in this study were integrated onto this surface using a copper-catalyzed azide-alkyne cycloaddition “click” reaction (CuAAC). The PNA aptamer (300 nM) solution was mixed with copper (II) sulfate (5 mM), sodium ascorbate (20 mM), and bathophenanthroline disulfonic acid disodium salt (bath) (5 mM), and the final mixture was deposited on the modified gold chip and incubated overnight. Finally, the prism was gently washed, dried, and then placed inside the Affinité P4SPR instrument for SPR measurements. Different cTnI protein concentrations (5, 20, and 50 pM) were passed over the N<sub>3</sub>-PNA aptamer-coated chips for the sensing experiment (injection time - 10 min, flow rate - 30 µL min<sup>-1</sup>), and SPR sensorgrams for the cTnI binding kinetics were retrieved.

#### DNase treatment on DNA and N<sub>3</sub>-PNA aptamers

For the DNase treatment studies, DNA and N<sub>3</sub>-PNA based aptamers (DNA-40 and PNA-37) were immobilized onto SPR prisms and were further exposed to physiologically relevant DNase I concentrations (5, 10, 20 ng/mL, prepared in 1 × PBS-TMg) for 2 h, then the prisms were gently washed, dried, and then placed inside the Affinité P4SPR instrument for SPR measurements. Different cTnI protein concentrations of (5, 20, and 50 pM) were passed over the N<sub>3</sub>-PNA aptamer-coated chips for the sensing experiment (injection time - 10 min, flow rate -
30 µL min⁻¹), and SPR sensorgrams for the cTnI binding kinetics and RU-based sensitivity measurements were retrieved.

### Pyrene-PNA aptamers' immobilization

The pyrene-PNA (pyPNA) immobilization does not require "click" chemistry and only involves deposition and incubation steps. Longer incubation times can lead to better immobilization and increased PNA fixation. A solution of PNA aptamer (300 nM) in 1 × PBS was deposited onto a graphene-coated gold SPR prism (the graphene transfer process was carried out similarly to our earlier work) [38], and incubated for 65 h at 4°C. Following incubation, the chip was washed with 1 × PBS, air-dried, and then placed inside the SPR machine for measurements. A solution of 1 × PBS containing 0.05 % Tween-20 and 1 mM MgCl₂ was used as the running buffer for cTnI binding experiments. A similar concentration of cTnI protein, ranging from 5 to 50 pM, was used here as well for measuring the binding kinetics (flow rate of 30 µL min⁻¹).

### Surface plasmon resonance (SPR)

SPR measurements were performed on a portable P4SPR platform from Affinité Instruments, Canada. The SPR instrument contains four channels (referred to as A–D) that operate in parallel; one channel serves as a reference throughout each experiment. The detector exhibits a resolution of 1.34 nm full width at half maximum (FWHM). SPR curves were recorded using a running buffer of 1 × PBS (with 1 mM MgCl₂ and 0.05 % v/v Tween-20) at a flow rate of 30 µL min⁻¹. Surface modification of the gold-coated prims was performed as per the protocol mentioned earlier. After the surface modification, the prism was placed inside the SPR, and all the cTnI binding titrations were carried out in running buffer operated with a peristaltic pump (Masterflex Peristaltic Pump System) at room temperature. The raw sensorgrams were extracted from the Affinité Instruments ezControl Software as CSV files and plotted using GraphPad Prism v7 software (GraphPad, Boston, Massachusetts USA, www.graphpad.com). For the extraction of the kinetic parameter values ($K_D$, $k_{on}$, $k_{off}$), the SPR traces were fitted using the "association then dissociation" kinetics model (built into the Nonlinear regression: binding kinetics section) of GraphPad Prism v7 software wherein the concentrations specific to each ligand (Hot<sub>NM</sub>) and time for the start of dissociation ($Time_0$) was provided to the equation model.

From the cTnI binding resonance response of the SPR across different concentrations, the steady state RU is considered to derive the standard calibration curve (cTnI concentration vs. RU signal). From this curve, the limit of detection (LOD) is calculated by the following formula [50]: $LOD = LOB + 1.65\sigma$ (LOB: limit of blank; $\sigma$: standard deviation of the low concentration sample). Wherein the LOB = $\mu_B + 1.65\sigma_B$ ($\mu_B$: mean of blank; $\sigma_B$: standard deviation of blank).

### Molecular dynamics simulation

The sequence of the DNA-40 aptamer was first transcribed into an RNA sequence by replacing thymine with uracil. The 3D model of the single-stranded RNA was built using the RNAComposer web server [51] based on the secondary structure predicted with the mFold web server [52,53]. The resulting 3D RNA model was converted back into DNA by replacing uracil with thymine. This was done manually by adding a methyl to replace the hydrogen in position 5. Then, all riboses on the backbone were converted into 2-deoxyribose by removing the oxygen in the 2' position. All hydrogen atoms in the final structures were removed and re-added in their correct positions, according to the structures of the residues stored in the selected force field (FF), during the preparation of topology files. Two independent simulations of the DNA strand were performed using two different FFs. The additive CHARMM36 FF [54], as distributed by MacKerell Lab, with DNA parameters published by Hart et al. [55]; and the Amber ff99 FF published by Kollman et al. [56], with the parmbsc0 [57] and OL15 correction [58] for DNA. These two FFs were selected to ensure consistency with those used to simulate PNAS

(see above). Topology generation and pairing with the FF parameters followed FF-specific workflows. For Amber FF, LEaP (AmberTools) [59] was used to prepare the parameter files, which were then converted to GROMACS format with ParmEd [60]. For CHARMM, topologies were prepared directly in GROMACS 2025.1 [61]. The GROMACS 2025.1 software package was used in both cases to solvate the strand, add ions and run the simulations.

The 3D model of the aptamer PNA-40 was prepared by modifying the structure file of DNA-40. The 2-deoxyribose-phosphate units backbone was converted into N-(2-aminoethyl)glycine units. The hemiacetal oxygen of the ribose and the oxygen 1 of the phosphate group was removed; the phosphorus was replaced with a carbon, the oxygen in 5' position and the carbon in 3' position of the ribose were replaced with two nitrogen and the oxygen in 3' position was replaced with carbon. The hydrogen bonded to the oxygen at the 5' position on the 5' end of the chain was replaced with an acetyl group using the GaussView 6 software [62]. This is necessary because the FFs require the nitrogen at the N-terminal end to be acetylated. All atoms of the PNA backbone have been renamed according to the residues present in the FF. All hydrogen atoms in the final structures were removed and re-added in their correct positions, according to the structures of the residues stored in FF, during the preparation of topology files. Two independent simulations of the PNA strand were performed using two different FFs published by Jasiński et al. [31], which provided optimized backbone torsion parameters for PNA for both the additive CHARMM36 and Amber FFs. To our knowledge, these are the most recent parameters that were optimized for PNA. As with DNA, the CHARMM force field, including these PNA parameters, is available in GROMACS format, so topologies can be prepared directly in GROMACS. In contrast, for Amber, parameters must be built with LEaP and then converted using ParmEd.

For the Molecular Dynamics Simulation, each system, comprising either a PNA or DNA aptamer, was solvated with TIP3P water molecules [63] within a dodecahedral box, maintaining a distance of 1 nm between the strand and the edges of the box. A physiological salt concentration of 0.1 M NaCl was added, and for the DNA systems, an additional forty sodium ions were introduced as counterions to ensure charge neutrality. Periodic boundary conditions (PBCs) were applied in all spatial dimensions. For all geometry optimization, equilibration and simulation, GROMACS 2025.1 software was used. All bonds involving hydrogens were constrained using the LINCS algorithm [64], and a 1.0 nm cutoff was applied for short-range electrostatic and van der Waals interactions. Long-range electrostatic interactions were computed using the Particle Mesh Ewald (PME) method. The energy of each system was minimized using the steepest descent algorithm. For the PNA system only, because the starting structure was highly distorted, energy minimization was performed in two stages. First, an in-vacuo minimization was run with positional restraints on the nucleobases to allow relaxation of the PNA backbone. Second, the solvated system was minimized, following the same protocol used for the DNA strand. This was followed by equilibration in the NVT ensemble for 200 ps at 300 K using the V-Rescale thermostat [62] and then for 200 ps in the NPT ensemble at 1 atm pressure, employing the Parrinello-Rahman barostat [65]. Production simulations were conducted for 1000 ns using a 2-fs time step to integrate the equations of motion.

### Credit authorship contribution statement

**Manova Santhosh Yesupatham:** Validation. **BAGALE Rupali:** Validation, Methodology. **David Montaigne:** Resources, Formal analysis. **Christophe Ritzenthaler:** Writing – review & editing, Supervision, Funding acquisition. **Francesco Basini:** Writing – original draft, Validation, Methodology, Investigation, Data curation. **Subhankar Sahu:** Writing – original draft, Validation, Supervision, Software, Methodology, Formal analysis. **HAMBLI Abdellah:** Visualization, Validation, Methodology, Data curation. **Roberto Corradini:** Writing – review & editing, Resources, Investigation, Funding acquisition,
**Conceptualization.** **Wolfgang Knoll:** Writing – review & editing, Supervision. **Henri Happy:** Writing – review & editing, Resources. **Eloise Woitrain:** Resources, Formal analysis. **Sabine Szunerits:** Writing – original draft, Supervision, Resources, Investigation, Funding acquisition, Conceptualization. **Rabah Boukherroub:** Writing – review & editing.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sabine Szunerits reports financial support was provided by University of Lille. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

Financial supports from the Centre National de la Recherche Scientifique (CNRS), the University of Lille and Danube Private University (DPU) are acknowledged. This work has benefited from the equipment and framework of the COMP-R Initiative, funded by the ‘Departments of Excellence’ program of the Italian Ministry for University and Research (MUR, 2023–2027) for the Department of Chemistry, Life Sciences and Environmental Sustainability of the University of Parma. The work was carried out with co-financing from the European Union – Next Generation EU, MISSION 4, COMPONENT 2, “From research to business”, INVESTMENT 1.4, “Strengthening research facilities and creation of "National R&D Champions", relating to the project identified with code CN00000041, entitled "National Center for Gene Therapy and Drugs based on RNA Technology- project BAC POPNART CUP C93C22002780006. The TeachInParma Program is also acknowledged for sponsoring the supervision and collaborative work of WK at the University of Parma premises. Financial support from the Fédération Française de Cardiologie (FFC) is acknowledged. Support of the government of Lower Austria and Tyrol under the COMET Project « PI-SENS »(Personalized Medicine enabled by intelligent sensing systems) FFG grant no. FO 999915477 is acknowledged.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.nantod.2025.102957.

## Data availability

Data will be made available on request.

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