ARTICLE IN PRESS Article in Press Mechanistic insights into melanin-induced PCR inhibition and its NanoPCR-based mitigation Scientific Reports Received: 16 July 2025 Accepted: 1 January 2026 Cite this article as: Vajpayee K., Srivastava S., Sharma S. et al. Mechanistic insights into melanin- induced PCR inhibition and its NanoPCR- based mitigation. Sci Rep (2026). https:// doi.org/10.1038/s41598-026-35010-w Kamayani Vajpayee, Shriyansh Srivastava, Shivkant Sharma, Swadha Gupta, Ashutosh Srivastava, Vidhi Paida, Hirak Ranjan Dash, Anju Pappachan & Ritesh K. Shukla We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply. If this paper is publishing under a Transparent Peer Review model then Peer Review reports will publish with the final article. https://doi.org/10.1038/s41598-026-35010-w © The Author(s) 2026. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. ARTICLE IN PRESS Mechanistic Insights into Melanin-Induced PCR Inhibition and Its NanoPCR-Based Mitigation Kamayani Vajpayeea, Shriyansh Srivastavab, Shivkant Sharmac, Swadha Guptad, Ashutosh Srivastavab, Vidhi Paidaa, Hirak Ranjan Dashe, Anju Pappachand, Ritesh K Shuklaaf aBiological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Central Campus, Navrangpura-Ahmedabad, Gujarat, 380009, India. bDepartment of Biological Sciences and Engineering, Indian Institute of Technology Gandhinagar, Gandhinagar, Gujarat, 382055, India cResearch Scientist, Ingenomics Pvt. Ltd, Delhi, India dSchool of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India eSchool of Forensic Science, Centurion University of Technology and Management, Bhubaneshwar, Orissa, India fDepartment of Forensic Science, National Forensic Sciences University, Raipur, Chhattisgarh, India Corresponding Author: Ritesh K Shukla Ritesh K Shukla, Associate Professor, Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University, Central Campus, Navrangpura-Ahmedabad, Gujarat, 380009, India. Department of Forensic Science, National Forensic Sciences University, Raipur, Chhattisgarh, India Email: ritesh.shukla@nfsu.ac.in Abstract Melanin is a potent inhibitor of PCR, which interferes with forensic DNA typing by binding to Taq polymerase and disrupting its function, leading to allele dropout and decreased peak heights. Traditional mitigation measures, such as dilution and replacement of the polymerase, often result in the loss of DNA and are not suitable for low-template forensic samples. In this study, melanin- induced inhibition was explored using molecular docking and dynamics simulation, which ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS identified stable interaction with catalytic residues TYR671 and PHE667 (Kd = 31.76 ± 0.02 µM), thereby interfering with polymerase function. STR typing of the inhibitor-treated sample showed a total allelic loss of SE33 and Penta E, dropout at D12S391, and substantially reduced peak heights. The overall peak height was 443,409.3 RFU. Facilitating methods using AuNPs, BSA- coated AuNPs, and standard BSA were further compared. AuNPs provided partial restoration (TPH = 545,605.7 RFU), whereas BSA-coated AuNPs provided improved restoration (TPH = 682,938.3 RFU) with harmonious heterozygous peaks (mean PHR = 0.90). Standard BSA had the highest restoration (TPH = 786,122.7 RFU; mean PHR = 0.92), restored alleles between dye channels. Statistical analysis revealed significant enhancement by BSA (p < 0.0001) and moderate enhancement by BSA-coated AuNPs (p = 0.0186). The BSA yielded optimal results, but it also exhibited larger sample differences. On the other hand, BSA-coated AuNPs offered more consistent facilitation at a very low concentration compared to BSA. This study thus explains the mode of PCR inhibition by melanin. It further demonstrates that BSA remains the most effective facilitator, but it also has its own limitations. In contrast, BSA-coated AuNPs offer a reliable nanotechnology-based method to overcome this limitation in forensic PCR applications. Keywords: Melanin, Gold nanoparticles, BSA, BSA-coated gold nanoparticles, PCR inhibition. 1. Introduction: The polymerase chain reaction, or PCR, is a scientific breakthrough invented in 1983 by Kary Mullis, for which he was awarded the Nobel Prize in Chemistry in 1993 [1]. As a vital technique in several scientific domains, including genetics [2-5], molecular biology [5- 7], and forensic science [8, 9]PCR can amplify small amounts of DNA into quantities suitable for many analytical methods [10, 11]. Despite its revolutionary potential and robustness, the value of PCR is often undermined by inhibitory compounds usually coextracted with DNA, especially in challenging samples. [12]. These compounds interfere with the DNA polymerase, the template, or the reaction kinetics. [12]. Among the most potent and forensically relevant inhibitors, melanin is a polyphenolic biopolymer found in biological materials such as hair, skin, and decomposing tissue [13, 14]. In forensic casework, samples derived from hair, highly pigmented tissues, cremated remains, or degraded biological materials contain an appreciable amount of melanin, with spectroscopic measurements ranging from 2–4% of the gross hair mass, as a function of colour and race [15, 16]. Though a seemingly small proportion of total hair mass, melanin's structural characteristics promote non-specific protein interactions, divalent metal ion sequestration such as Mg²⁺, and hindrance to DNA template binding, all of which can reduce the efficacy of DNA polymerase ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS [17]. Eckhart et al. (2000) [18] demonstrated that melanin has reversible binding with thermostable DNA polymerase, reducing its activity at concentrations below 200 ng/mL, which can be mitigated through dilution or the addition of bovine serum albumin. Suenaga and Nakamura [19] suggested that DNA from pigmented hairs contained inhibitors tied to the pigment that caused failures in amplification during the use of the ISOHAIR method, while use of the Chelex and QIAamp extractions eliminated the effect. Other studies have confirmed that both natural and synthetic eumelanin inhibit mitochondrial as well as nuclear DNA fragment amplification at microgram-per-millilitre quantities [18, 20]. Thompson et al. [21] further showed that melanin decreases amplification efficiency and allele recovery during short tandem repeat (STR) typing. A recent study on pigmented tissue suggested that co-purified melanin inhibits multiplex PCR unless special procedures are applied to remove the inhibitors [22]. Together, these findings emphasise that while melanin will rarely pose a significant problem in standard casework, its presence in pigmented or damaged samples becomes a considerable hindrance to reliable DNA profiling. For forensic casework purposes, samples from hair shafts, pigmented tissues, cremated or decomposed materials may yield incomplete or failed amplification of PCR [23]. Inhibition of this type results in partial or no generation of STR profiles, allele substitution and reduction of peak height with severe impairment of evidentiary quality in a forensic setting [24]. Although well-documented inhibitory effects, the molecular mechanism by which melanin suppresses DNA amplification remains unknown. Several hypotheses have been proposed, suggesting that melanin either directly interacts with Taq polymerase, causing structural changes that reduce enzymatic activity [18, 23] or the redox-active properties of melanin may generate reactive oxygen species (ROS), which cause oxidative damage to DNA and polymerase inhibition [25]. However, none of these mechanisms has been conclusively demonstrated, leaving a critical gap in DNA analysis. Structurally, melanin is classified into three broad types: eumelanin, pheomelanin, and neuromelanin. The most common type, eumelanin, produces black and brown pigmentation. [26]. It mainly comprises 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) subunits, forming an insoluble and stable polymer. [26]. Forensic evidence from melanin-rich biological tissues, such as hair, skin, and charred remains, presents significant issues in DNA analysis. Given the importance of reliable DNA amplification in forensic analysis, elucidating and overcoming melanin-induced PCR inhibition is imperative. [27]. Although efforts to overcome inhibition through dilution [28], the addition of BSA (bovine ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS serum albumin) [29, 30], and the use of alternative chemical additives has been made; these approaches often compromise sensitivity or result in incomplete DNA profiles. Notably, BSA has certain inherent disadvantages, despite its frequent use. Its effects differ among loci, templates, and polymerases, and it is highly concentration-dependent; in the absence of inhibitors, it may even reduce amplification efficiency [31, 32]. According to research, a BSA concentration of approximately 660 µg/ml is required to eliminate inhibition, which is impractically high [33]. Furthermore, even at lower but still significant doses (500 µg/ml), BSA has enhanced amplification while blocking primer–dimer formation [33]. Additionally, its wider use is limited by batch-to-batch variability [33, 34] and the potential for trace DNA or nuclease contamination [31]. Structural limitations further limit its functionality. Additionally, BSA can be eliminated through heat cycling, and after several cycles, the enabling effect of BSA can be eliminated by steric hindrance caused by ,significant inhibitors [31, 32]. Therefore, optimising the BSA concentration to the amplification process has been suggested. Both separately and in combination, these drawbacks emphasise the need for more robust, all- encompassing, and adaptable facilitators. Despite these limitations, the unique physicochemical properties of nanoparticles offer a practical means of more effectively addressing PCR inhibition. Accordingly, gold nanoparticles can be adjusted to improve their interaction with inhibitors while maintaining enzymatic activity due to their high surface-area-to-volume ratio, excellent biocompatibility, and customizable surface chemistry [35]. It has been proposed that the binding affinity and inhibitor resistance of AuNPs can be improved by coating them with BSA [36]. It is unlikely, nonetheless, that such BSA-coated AuNPs will be able to precisely and efficiently overcome melanin-induced inhibition without being constrained by concentration-dependent parameters, structural constraints, or the unpredictable nature of free BSA. To advance PCR facilitation procedures that are more robust, flexible, effective, and capable of handling complex forensic sample matrices, it is necessary to overcome this obstacle. The forensic community needs a more robust, potent, and targeted solution to overcome the failures of PCR caused by melanin. Addressing this issue will considerably improve the reliability and evidentiary value of forensic DNA typing by mitigating the inhibitory effect of melanin during amplification, especially in cases where heavily pigmented biological samples are the primary sources of evidence. In this regard, the present study aims to explore melanin as a forensically relevant PCR inhibitor, first investigating its interactions computationally and biophysically to determine possible melanin binding sites. Since the computational assays fundamentally make ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS assumptions that restrict their ability to observe protein–ligand interactions experimentally, and additionally, interactions with many residues—not all of which are fluorescently active—were suggested by the simulations. These results were further experimentally validated using the tryptophan fluorescence quenching assay, a sensitive reporter of conformational or microenvironmental changes near tryptophan residues. It then evaluates BSA-coated and bare gold nanoparticles based on their ability to improve melanin-mediated PCR inhibition. Although the specific site-directed binding assays were not performed in this study, the observation of an improved amplification efficiency provides functional evidence that BSA- coated and bare gold nanoparticles can modulate melanin–polymerase interactions. This combined approach-a synthesis of mechanistic insight and functional verification-affords a systems-level approach to resolving PCR inhibition and informs subsequent targeted manipulation. Thus, providing a mechanistic account of its inhibitory activity and proposing a possible strategy for overcoming its effect. By elucidating the molecular interactions of melanin with DNA polymerase, a critical enzyme in the amplification process, this study seeks to improve forensic DNA profiling methods. Determining a precise inhibition mechanism will not only advance the understanding of melanin's effect but also guide the development of more refined protocols for forensic laboratories, thus ensuring improved success rates in DNA amplification of melanin-containing forensic samples, especially hair and other pigmented tissues. 2. Results: 2.1. Molecular Docking and Molecular Dynamics Simulation: A critical study of Taq polymerase, a crucial component of the PCR, structure revealed that it has an N-terminal exonuclease domain (residues 1-289) and a C-terminal polymerase domain (residues 306- 832), which are connected by a short linker (Supplementary Figure S1A). The polymerase domain, also known as the Klentaq domain, is further subdivided into Finger, Palm, and Thumb subdomains. The catalytically crucial residues—ASP610, ASP785, and GLU786—are in the Palm subdomain, where they coordinate with Mg²⁺ for nucleotide incorporation. The Finger (helix O) and Thumb (helices I and H) subdomains are ancillary for polymerisation. The Thumb has additional conserved residues, ASN485, SER515, and LYS540, which interact with the minor grooves of DNA. Additional conserved residues with contact with DNA are ARG573, GLU655, TYR671, ASN750, GLN745, HIS784, and ASP785 (Supplementary Figure S1B). During amplification, Taq switches from the open ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS conformation (1TAQ) to the closed, DNA-bound conformation (3KTQ) (Supplementary Figure S1C and D) [37, 38]. Thus, based on the importance of the catalytic core, it was hypothesised that melanin could bind to the core and thereby suppress enzymatic activity. Molecular docking and dynamics simulations were employed to investigate the hypothesis using four structural models: truncated 1TAQ, 3KTQ, 3KTQΔDNA, and 3KTQΔDNAΔMg. In 1TAQ, melanin had moderate binding energy (–4.158 kcal/mol), interacting with ASP785 and GLU615 through salt bridges, ARG587 through hydrogen bonding, HIS784 through π–π stacking, and VAL586 through hydrophobic contacts (Supplementary Figure S3a). The 3KTQ complex had the highest binding (–6.442 kcal/mol). Melanin hydrogen-bonded with ARG677 and ARG587, salt-bridged with DG110 and DC111, and π–π stacked with DC109, positioning itself in the DNA helix (Supplementary Figure S3b). The DNA deletion from the crystal structure (3KTQΔDNA) decreased the binding affinity by 4.521 kcal/mol. Melanin, nonetheless, still engaged catalytically significant residues, including hydrogen bonding with GLU615 and GLN754. In the absence of DNA, hydrophobic interactions with MET673, TYR671, and PHE667 appeared (Supplementary Figure S3c). The docking score was further enhanced to –5.709 kcal/mol in structure 3KTQΔDNAΔMg. Melanin engaged ARG573 through hydrogen bonds, GLU615 through salt bridges, and PHE667 through π-cation interactions. Hydrophobic interactions with TYR671 and MET673 have also been observed (Supplementary Figure S3d). To investigate complex stability in greater detail, 50-ns MD simulations were conducted. RMSD values monitored conformational drift, and protein-ligand (PL) contact analysis found stabilising residues (Figure 1). In 1TAQ, RMSD varied from 1.5 to 10.5 Å, indicating instability. ASP785 and HIS784 dominated the initial interactions, while interaction by ARG573 was delayed. Transient contacts with ASN583 and HIS784 were observed (Figures 1B, 1F). 3KTQ displayed the lowest RMSD, ranging from 2 to 5.4 Å. Although melanin showed transient initial interactions with GLU507 (Figure 1C, 1G), its binding was not stable. Still, the DNA-bound form of the enzyme appears to stabilise the overall architecture even if melanin's anchorage is poor. 3KTQΔDNA and 3KTQΔDNAΔMg had increased RMSD values with 6–16 Å and 1.5–10 Å, respectively. Stable hydrophobic contact with PHE667 and TYR671 in 3KTQΔDNA was maintained throughout the simulation (Figures 1D, 1H). GLU615, PHE667, and TYR671 in 3KTQΔDNAΔMg regularly contacted melanin (Figures 1E, 1I). ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS Figure 1: Figure panel illustrating the results of molecular dynamics simulations and protein-ligand interaction profiles of melanin-bound Taq polymerase complexes. (A) Root Mean Square Deviation (RMSD) in Åwas calculated for four different protein-melanin complexes, and colour was coded as: blue: 1TAQ, orange: 3KTQ, purple: 3KTQΔDNA, and brown: 3KTQΔDNAΔMg. (B-E) The ligand-protein contact diagram simulation. Here, B: 1TAQ; C: 3KTQ; D: 3KTQΔDNA; E: 3KTQΔDNAΔMg. (F-I) Protein-ligand interaction contact bar plots for the four complexes, quantifying the cumulative interaction frequencies between melanin and individual residues of the protein across the entire simulation. Here, F: 1TAQ, G: 3KTQ, H: 3KTQΔDNA, and I: 3KTQΔDNAΔMg. 2.2. Tryptophan Quenching Assay: The saturation binding experiment was performed using tryptophan fluorescence determination, and the Kd value for melanin was found to be 31.76 ± 0.02µM (Figure 2). Figure 2: Tryptophan quenching analysis of protein in the presence of increasing melanin concentrations. A: Stern- Volmer plot showing the quenching efficiency of tryptophan residues. B: Emission spectra of tryptophan fluorescence at increasing concentrations of melanin (0-100 µM). 2.3. Nanoparticle Characterization: Successful synthesis and functionalization of AuNPs and BSA-coated AuNPs were verified using UV-visible spectroscopy (SPR band at ~520 nm red shifting to ~525–530 nm for BSA-coated particles), dynamic light scattering (hydrodynamic diameter: 33.07 nm for AuNPs; 47.68 nm for BSA-coated), zeta potential (-29.1 mV to -15.2 mV), TEM imaging (14 nm and 26 nm, respectively), and FT-IR ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS spectroscopy (presence of characteristic amide I and II bands at 1648 cm⁻¹ and 1549 cm⁻¹) (Figure 3). According to studies, BSA forms a protective monolayer over AuNPs, and stabilisation is accomplished through interactions between protein side chains and domains that reduce solvation enthalpy and entropy. This stops aggregation and ensures colloidal stability. [39]. Long-term stability is further enhanced by the constant exchange of free BSA in solution with BSA molecules adsorbed onto the nanoparticle surface. Crucially, AuNP aggregation occurs when the concentration of BSA falls below the critical threshold of approximately 30 nmol/L, or about 23 BSA molecules per nanoparticle. This is sufficient to stabilise the nanoparticle, but less than what is required for maximum monolayer coverage saturation. A saturation monolayer coverage of 14 nm bare AuNPs is achieved by adsorption of no more than about 50 BSA molecules. [39]. Therefore, it was anticipated that the BSA-coated AuNPs developed in this work would function as a stable colloidal system with sub-monolayer to near-monolayer coverage, the least amount of aggregation, and the highest reproducibility in terms of future utility. According to research by Shi et al. (2012), the quantitative concentration of AuNP stock was 3.18 nM (nanoMolars), with almost 30 BSA molecules adsorbed per nanoparticle. [39]. The final PCR reaction, which had a capacity of 10 µL and contained 3 µL of AuNPs and BSA-coated AuNPs stock, revealed that the effective concentration of AuNPs was 0.954 nM, corresponding to 5.75×10¹¹ particles/mL. This indicated that 18.9 ng of BSA per reaction, or approximately 1.72 × 10¹³ BSA molecules/mL, were adsorbed. Interestingly, this concentration is many times lower than the BSA control concentration of 1 mg/mL (4.51µM), which is typically used as a parallel facilitator. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS Figure 3: Characterisation of the synthesised bare AuNPs and Au: BSA. A) & B)- Representative TEM images (normalized to scale 200nm) of bare AuNPs (14 nm) and Au: BSA NPs (26 nm) respectively; C) Zeta Potential of the bare and BSA coated Gold nanoparticles in mV D) FTIR spectra recorded from Bare AuNPs (green), Au: BSA (Blue), and BSA (red) show the presence of Amide I and Amide II bands at 1648 cm⁻¹ and 1549 cm⁻¹; ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS respectively and E) UV spectra recorded from bare AuNPs and Au: BSA at 520 nms and the shift in the peak due to BSA coating. 2.4.nanoPCR-based STR genotyping: Melanin-treated samples had degraded STR profiles with no SE33 and Penta E amplification and partial dropout at D12S391 (Figure 4). Some of the loci, such as D21S11, D7S820, CSF1PO, Penta D, D2S441, D8S1179, FGA, DYS391, and D10S1248, had severe RFU reduction. The total peak height (TPH) of melanin-suppressed profiles was 443409.3 RFU. D12S391 and FGA had low PHRs (0,0.8,0.77, and 0.78,0.78,0.78) respectively, but the majority of the loci had PHRs averaging 0.88, with TPOX (0.98), D19S433 (0.99), and D10S1248 (0.98) being close to balance. The local balance mean was 0.92. For the dye channels, violet had the minimum signal (17266 RFU), while blue (124885 RFU), black (116313 RFU), green (92737 RFU), and red (92208 RFU) had relatively higher intensities, suggesting differential quenching by melanin. The addition of Facilitator 1 (AuNPs) partially recovered the STR profile. Penta E and SE33 showed re-emergent peaks (4,196 RFU and ~229 RFU, respectively), and TPH was raised to 545605.7 RFU. Fluorescence was enhanced in all violet, blue, black, green, and red channels: 48305 RFU, 114528 RFU, 167717 RFU, 99487 RFU, and 115568 RFU, respectively. Mean PHR was raised to 1, with FGA (0.96,0.96,0.96), D12S391(0.79,0.79,0.79), and SE33 (0.99,0.99,0.97) being recovered, respectively. The mean local balance was increased to 0.93. Although there was an improvement in the profile, AuNPs were unable to completely abolish the inhibitory effect of melanin. However, even locus-wise uniform improvement (absence of ski-slope) was observed. Facilitator 2 (BSA-coated AuNPs) further improved the profile. Penta E (4,519 RFU) and SE33 (382, 384 RFU) had better recovery strength. TPH increased to 682938.3 RFU. Dye channel fluorescence improved to: blue (167536 RFU), black (196755 RFU), green (136930 RFU), red (113603 RFU), and violet (52950 RFU). PHRs for FGA (0.79,0.80,0.79), SE33 (1,0.99,0.99) and D12S391(0.97,0.98,0.95) were also improved further. Overall, PHR averaged 0.90, with a mean local balance of 0.93. Treatment with conventionally used BSA resulted in the highest degree of STR profile recovery. The TPH increased exponentially to 786122.7 RFU, significantly surpassing levels attained with BSA-coated AuNPs. Allelic balance was also efficiently restored, with the PHRs on average being 0.92 across loci. Dye channel signals also improved with blue, black, green, red, and violet dye channels having 176343.7 RFU, 233229 RFU,167041 RFU, 128216.7 RFU, and 81292.3 RFU, respectively. The MLB increased to 0.95. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS 2.5. Statistical Analysis: Statistical evaluation of amplification performance demonstrated clear differences in the efficacy of facilitators. A Kruskal–Wallis test demonstrated a significant overall effect of the treatment on total peak height (TPH) (H = 52.31, p < 0.0001). Post-hoc analysis demonstrated that neither AuNPs nor AuNP–BSA influenced amplification in uninhibited controls (p > 0.05), while BSA significantly increased TPH relative to controls (p < 0.0001). In melanin-inhibited samples, AuNPs again conferred no benefit (p > 0.9999), while both BSA and AuNP–BSA significantly restored amplification (p = 0.0001, p = 0.0186), with BSA producing the greatest magnitude of recovery. Allelic balance was not affected much by any of the treatment conditions. The PHR values only indicated a small overall group difference: H = 9.75, p = 0.0448; adjusted pairwise comparisons did not indicate any statistically significant deviations from the untreated control, thus indicating no allelic distortion due to facilitators. Channel-specific analysis demonstrated significant improvements in the selected dye channels, especially under melanin inhibition. BSA showed consistent enhancement of signal intensities across multiple channels, while AuNP–BSA demonstrated notable recovery especially in the blue and black channels (p < 0.01). AuNPs did not improve amplification in any channel tested. In the direct comparison of facilitators, there was no significant difference in TPH or PHR between BSA and AuNP–BSA; p > 0.15 (Supplementary Table S3h). However, the amplification with AuNP–BSA was markedly less variable, CV 16– 18%, compared to free BSA, CV 22–24%, suggesting a more homogeneous amplification response and an improved reproducibility. Complete statistical metrics, including effect sizes, Hodges–Lehmann estimates, and channel-wise comparisons can be found in Supplementary Table S3 (Supplementary Table S3h). Analysis of the CV showed wider variability in the efficiency of amplification between BSA-treated samples compared to between AuNP–BSA. BSA samples had wider variability (CV for time intervals for amplification of 22–24) than AuNP–BSA samples that closely clustered together (CV between 16–18), which had less variability between samples (See Raw data file, sheet 1-Analysis). ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS Figure 4: A Box plot depicts selected STR markers that showed allele drop and reduction in RFU upon treatment with melanin, which was subsequently improved following facilitator treatment. For heterozygous markers, both alleles were individually analysed to assess the statistically significant impact of the treatments. 3. Discussion: The fundamental role of PCR in forensic DNA analysis and molecular diagnostics is supported by its dependability. Nevertheless, co-extracted inhibitors continue to be a significant barrier to adequate amplification, especially in low-template or degraded samples ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS [12]. Of these, melanin has been consistently reported as a strong PCR inhibitor and is relatively abundant in pigmented clinical and forensic samples [24]. Nevertheless, there are clear descriptions of its inhibitory effects; however, there has been insufficient knowledge of the mechanism underlying it. Through this action, this work offers a comprehensive explanation at the molecular level of the inhibition of PCR caused by melanin, involving docking, MD, binding experiments, and profiling of STR. Structural examination of Taq polymerase revealed a catalytic Palm subdomain (residues ASP610, ASP785, GLU786) coordinated with Mg²⁺ for nucleotide incorporation, and DNA-contacting residues (PHE667, TYR671, GLU615) that stabilize polymerization. Docking showed that melanin anchors preferentially to these residues, particularly in the DNA-free and Mg²⁺-depleted forms of Taq, where accessibility to catalytic pockets is higher. Melanin bound with moderate affinity (Kd = 31.76 µM) through non-covalent interactions, including salt bridges, π–π stacking, and hydrophobic contacts, disrupting both template binding and catalysis. MD simulations confirmed that melanin destabilises the enzyme conformation, with persistent anchorage to PHE667 and TYR671, residues critical for nucleotide stabilisation and DNA positioning. Collectively, these results demonstrate that melanin is a mixed-mode, competitive, reversible, moderate-strength inhibitor of Taq polymerase, interacting non-covalently with the polymerase's catalytic residues and DNA binding groove, thereby influencing both polymerase activity and template accessibility. Previous studies have either described the empirical trends of inhibition or speculated that intercalation of DNA is the principal pathway [12, 24, 40-42]. In particular, McCord and colleagues described melanin as a type of DNA-binding inhibitor that demonstrated template masking and competitive binding, but failed to define particular molecular targets [43]. Remarkably, the present study employs computational methods and identifies that melanin anchors to PHE667, TYR671, and GLU615, which are critical residues that support the polymerase structure, stabilise nucleotides, and elongate the strand. These interactions persisted in different conformations, particularly in the presence of Mg2+ or in DNA-free conditions, implying that melanin can unfavourably destabilise the structural architecture of an enzyme even in the absence of DNA. Simulation outputs are also consistent with the data of fluorescence quenching, which proves a medium binding affinity of melanin (Kd = 31.76 μM) and the static, non-collisional nature of the interaction with the enzyme [44]. Other previous findings based on STR are also supported by our data and even mechanistically elucidated. Allelic dropout, a decrease in the rate of RFU, and imbalance between signals were ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS jointly linked with melanin inhibition, as reported by Funes-Huacca et al. [45] and Sidstedt et al. [46], who also detected signal impairment in melanin-contaminated forensic samples, which were also corroborated in this study. Consistent with these observations and the present study's structural findings, STR profiling revealed locus-specific allelic dropout, reduced total peak heights, and heterozygote imbalance, with occasional stochastic increases in RFU at certain loci, reflecting the non-linear and context- dependent nature of inhibition. High dropout rates at discriminatory loci such as SE33 and Penta E highlight the forensic implications of melanin contamination, which can erode identity resolution and complicate mixture interpretation. Specifically, the dropout at SE33 and Penta E loci, considered to possess high discrimination power, is a major concern in case work, where loss of these markers may degrade the identity resolutions or kinship inferencing. [47, 48]. In addition to this, disproportionately high heterozygote peaks at D12S391 and FGA are typical of genotyping a degraded sample and indicate severe consequences to mixed-profile interpretation. [49, 50]. Notably, in some cases, the inhibited samples had higher RFU levels than their corresponding controls, initially suggesting counterintuitive results. This apparent disparity may be explained by the stochastic nature of PCR in inhibitory conditions. Melanin does not have an equal inhibitory influence upon polymerase activity throughout all loci; instead, it acts in locus-specific and amplicon length and primer-dependent (efficiency) fashion. Therefore, while most loci exhibited the expected strong reduction in intensity signal, some loci had relatively elevated RFU levels, likely reflecting the sporadic occurrence of amplicons or partial alleviation of inhibition in selected reactions. Such apparent differences align with the multifaceted and nonlinear effects of inhibitors on PCR kinetics, highlighting that RFU reductions must be assessed in the context of overall image quality and the balance among peaks, rather than by absolute measures. Moreover, under low- template or inhibited PCR conditions, stochastic effects associated with sampling variation during early amplification cycles can result in certain loci showing disproportionate gain in signal. Such stochastic effects are well understood in forensic genotyping, especially for alleles with shorter amplicon lengths or superior primer binding kinetics. Therefore, the occasionally high RFU values do not indicate improved amplification performance but reflect stochastic PCR kinetics under inhibitory stress. To circumvent this issue, nano-based mitigation approaches were investigated through bare AuNPs, BSA, and BSA-conjugated AuNPs. Bare AuNPs failed to recover amplification, and BSA ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS exhibited robust and consistent recovery throughout all dye channels, verifying it as a universal facilitator. Importantly, Au:BSA nanoparticles achieved comparable signal strength and allelic balance recovery to BSA, with enhancements in specific channels. Although BSA possessed greater peak heights, examination revealed that Au:BSA ensured greater uniformity with narrower amplification distributions throughout the samples. This indicates that Au:BSA provides uniformity in amplified products, although BSA contains higher raw signal levels. The increased consistency afforded by BSA-AuNPs likely stems from the nanoparticle scaffold serving as a thermally and conformationally stabilizing surface for BSA. Our characterisation data support that conjugation to AuNPs enhances colloidal stability and reduces aggregation of BSA, maintaining its binding interface for melanin through thermocycling. This immobilisation offers a more homogeneous biochemical milieu during amplification, diminishing replicate-to-replicate variability and lessening stochastic inhibitory effects on Taq polymerase. Even though the greater quantity of BSA produced a more thorough recovery of amplification signals, BSA-coated gold nanoparticle formulations remained to offer measurable advantages, especially in stabilizing amplification where large amounts of the reagent are inappropriate. Higher reagent concentrations are more expensive, impair reaction stability, and probably conflict with workflows that have limited volume or resources. Thus, BSA-coated AuNPs could be used as low- dose (0.954 nM AuNPs with 18.9 ng BSA) nanotechnology-supported substitutes, ready to facilitate long-term PCR. As little as 18.9 ng of BSA, several orders of magnitude lower than that of ordinary BSA, coated onto the AuNPs successfully alleviates melanin-induced inhibition by promoting surface modification and higher-order bioavailability, supporting its efficacy and precise mediation. While dilution or post-extraction cleaning schemes lose DNA and suffer allele-specific errors or are not compatible with forensic STR platforms, this in situ nano-enabled approach recovers amplification without DNA loss and remains compatible with forensic STR platforms without allele-specific errors. Au:BSA's dual functionality as both inhibitor scavenger and enzyme- template stabilizer provides one additional benefit over classical facilitators. These results align with previous findings that BSA interacts with melanin through hydrophobic and π–π stacking interactions but demonstrate nanoparticle conjugation enhances consistency and robustness under inhibitory conditions [51]. The proposed Au:BSA NPs demonstrated high recovery in terms of TPH, fluorescence, and allelic balance, with statistically significant findings that underscore the superiority of Au:BSA over bare AuNPs. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS The outcomes of this study can be compared to conventional strategies such as dilution, chelation, or enzyme replacement, which typically lead to template loss and incomplete recovery as reported by Hu et al. and Vicente et al [52, 53]. This is an in situ clean-up method, in contrast to conventional post-extraction clean-up methods that involve pre-treatment, which can result in the loss of DNA. The fact that peak height ratios could be reconstituted without the production of allelic artifacts also establishes that this type of nanoPCR system should be compatible with conventional forensic genotyping platforms. The improvements in global dye fluorescence were also observed, with high signal rates in largely inhibited channels, supporting the usefulness of Au:BSA in reclaiming reaction efficiency without instating biased amplification kinetics. This work therefore, offers a conceptual breakthrough in inhibitor mitigation by combining molecular modeling with validation. Prior research has documented the use of nanoparticles to improve PCR; however, these studies lacked mechanistic insight and selectivity, frequently failing in complex matrices. With a broader potential for low-quality samples, the presented dual-mode platform,which combines selective scavenging and enzyme protection,represents a matrix- compatible and mechanistically sound approach. This approach also provides an effective weapon to address contaminated, decomposed, or pigmented samples in forensic genetics. Moreover, it is non-destructive and scalable, thus significantly broadening its use to cover archaeogenetics, wildlife forensics, and environmental DNA (which sometimes require co-purifying inhibitors such as melanin, humic acids, and bile salts). The nanoparticle platform has opportunities for future expansion in terms of modularity. Surface functionalization would allow broad-spectrum PCR facilitators to be specific to other inhibitors. Its robustness will also be tested within the full context of field testing on real forensic samples, saliva, bone, and mixed stains. BSA-coated AuNPs are innovative facilitators with significant applications in point-of-care diagnostics, forensic DNA typing, and other fields, owing to their low dose requirement, target-assisted functionality, and eco-friendly green design. Although this study demonstrated that BSA-coated AuNPs can act as facilitators and provide mechanistic insights into melanin-mediated PCR inhibition, it is essential to acknowledge certain limitations. In the present study, a single inhibitor was used in well-monitored laboratory settings. The inherent heterogeneity in forensic materials, such as fluctuations in inhibitor composition, concentration, and matrix complexity, may have different effects on facilitator efficacy than those observed here. Future work will involve validation using melanin-rich forensic samples, including shed hair, skin tissue, and casework extracts where inhibitor composition and matrix effects are ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS more variable. Such studies are essential for evaluating the robustness, scalability, and forensic admissibility of this nanoenabled mitigation strategy. Moreover, although the mechanistic investigation revealed potential sites of polymerase-melanin binding interaction that the present study did not experimentally verify as being overrun by BSA-coated AuNPs, there is a need for additional studies carried out under realistic forensic scenarios for the purpose of validating the strength, scalability, and mechanistic efficiency of the hypothesised mitigation mechanisms. 4. Methods 4.1. Materials: DNA Standards: Control DNA (9948A; 2ng/µl) was procured from Ingenomics™ Pvt Ltd, India, and was used to generate the control (inhibitor-free) DNA STR profiles. Taq DNA polymerase (Cat. MB101-0500) was purchased from BioHelix Co. Ltd. (Taiwan). Inhibitor: Melanin (inhibitor) and ammonium hydroxide (specific gravity 0.91) were purchased from Sigma Aldrich, US, and RFCL, Ltd, New Delhi, India, respectively. Nanoparticle (Facilitator) Synthesis: Sigma Aldrich, USA, purchased chloroauric acid (HAuCl4). Trisodium citrate (Na3C6H5O7) (minimum assay 98%) and Bovine Serum Albumin (BSA) (minimum assay 98%) were obtained from Hi-media (Mumbai, India). It was used as the nanoparticle surface modifier and control facilitator. Milli-Q ultrapure water was used to make and reconstitute all the reagents. Genotyping: Ingenomics™ AutoProfiler STR Kit (Ingenomics Private Limited, India) was used for amplification and genotyping of the control, melanin-inhibited, and facilitator- treated samples. The kit is based on 6-dye chemistry and includes 24 Autosomal STRs (Short Tandem Repeats), 1 Y-STR, an Amelogenin marker, and 2 Y-indels (Rs2032678; Rs771783753) All the statistical analyses were done using GraphPad Prism software (v8.0.2), GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com. 4.2. Structure-based modelling and interaction analysis:4.2.1. Curation of crystal structure and small molecule data: All the crystal structures corresponding to Taq polymerase (Uniprot: P19821) were retrieved from the Protein Data Bank (PDB). Two crystal structures corresponding to the open and closed conformations of Taq polymerase ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS (1TAQ and 3KTQ, respectively) were selected for molecular docking and molecular dynamics simulation. The structure data file (.sdf) for melanin was retrieved from PubChem (PubChem ID: 6325610) (Supplementary Figure S2). Details for crystal structures (1TAQ and 3KTQ) data are provided in Supplementary Figure S1A and C. 4.2.2. Molecular Docking and Molecular Dynamics Simulations: Molecular Docking was carried out using the Glide module of Schrödinger (Schrödinger Release 2022-3: Maestro, Schrödinger, LLC, New York, NY, 2022). The protein structures corresponding to 1TAQ and 3KTQ were downloaded, and the structure was processed using the Protein Preparation module (ProteinPrep) (Protein Preparation Workflow; Epik, Schrödinger, LLC, New York, NY, 2022; Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger, LLC, New York, NY, 2023) with default parameters. Since the closed structure, 3KTQ lacks the exonuclease domain, the corresponding exonuclease domain (residues 1-290) in the 1TAQ structure was removed, as mentioned by Nedumpully et al. [54]. The 3KTQ protein structure was further prepared to generate three different crystal states: 3KTQ, 3KTQ without DNA (3KTQΔDNA), and 3KTQ without DNA and Mg+2 ions (3KTQΔDNAΔMg). These four different models for Taq polymerase were used for docking studies. The Taq polymerase active site residues identified from a previous study by Eom et al. [38] were used for docking grid generation with the Receptor-Grid Generation module, ensuring accurate placement of melanin in the binding pocket (Supplementary Table S1). Lastly, melanin was preprocessed using the Ligand Preparation module (LigPrep) (Schrödinger Release 2022-23: LigPrep, Schrödinger, LLC, New York, NY, 2023) and further docked to protein structure models (1TAQ, 3KTQ, 3KTQΔDNA, 3KTQΔDNAΔMg) in extra precision mode (XP docking) using the Glide module (Schrödinger Release 2022-3: Glide, Schrödinger, LLC, New York, NY, 2022). The docking scores were used to evaluate the binding affinities of melanin to different Taq polymerase models. Molecular dynamics (MD) simulations were performed on the docked complexes to evaluate the stability and dynamic behaviour of the melanin–Taq polymerase interactions observed through docking. The melanin-docked protein complexes were then used to perform molecular dynamics (MD) simulations. All the simulations were performed with an OPLS4 force field [55] using the Desmond module by Schrödinger (Schrödinger Release 2022-3: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2022, Maestro- ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS Desmond Interoperability Tools, Schrödinger, New York, NY, 2022). The simulation system was briefly prepared using the System Builder tool as an orthorhombic box with TIP3P water as the explicit solvent and Na+ and Cl- ions to neutralise the system. The system was energy minimised and equilibrated with the default settings in the Molecular Dynamics tool (Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2022; Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2022.), which includes restrained and unrestrained stages under NVT and NPT ensembles. This involves gradual heating to 300 K and pressure stabilization at 1 atm using the Nosé– Hoover thermostat and Martyna–Tobias–Klein barostat [56]. The production run was performed using an NPT ensemble at 1.01325 bar pressure and 300K temperature for 50 nanoseconds (ns) with the option to relax the system before starting the MD simulation. The trajectories for each simulation system were analyzed using the Simulation Interaction Diagram tool (Desmond module) (Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2022. Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2022.), with melanin selected as the ligand. 4.2.3. Tryptophan Quenching Assay: To complement and validate molecular interactions observed through molecular docking and MD simulations study, fluorescence quenching experiments were performed using a Shimadzu fluorescence spectrophotometer at 25°C. This approach aimed to study the interaction between Taq polymerase and melanin, which could be corroborated by monitoring the changes in the intrinsic fluorescence of Taq polymerase upon binding to melanin. The samples were excited at 280nm, and emission was recorded between 290 and 400nm, with a 5nm slit width. To determine the binding strength of melanin to Taq polymerase, 15–20 aliquots of melanin from a 3.4mM stock solution were titrated against a fixed concentration of 0.1μM Taq polymerase. As melanin was introduced, changes in fluorescence were measured, and the possible binding was confirmed. The quenching assay was done in triplicate. The dissociation constant (Kd), which is a measure of binding, was calculated using the standard binding equation: Y = Bmax × [X] [X] + Kd Where Y represents the measured fluorescence signal, Bmax is the maximum binding capacity, [X] is the concentration of melanin, and Kd is the dissociation constant. 4.2.4. Inhibitor and Facilitator Preparation: A 1mg/ml stock solution of melanin was prepared in 0.5 N ammonium hydroxide, which was used as an inhibitor for the present ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS study. Gold nanoparticles (AuNPs) were synthesized using the method elucidated by Enustun and Turkevich, where 1 mM of 45 mL HAuCl4.3H2O was reduced by using 38.8 mM of 5 mL freshly prepared trisodium citrate (TSC) [57]. The synthesised nanoparticles are citrated gold nanoparticles, also known as AuNPs/ Bare gold nanoparticles. In the present study, AuNPs were considered Facilitator 1 for the PCR reaction. As Facilitator 2, AuNPs coated with BSA (Au:BSA) were synthesised, where 1mg/mL BSA was incubated with the AuNPs. Unbound and free BSA was removed by centrifugation (10,000 revolutions per minute for 15 mins). The pellets were resuspended in Milli-Q water. The efficacy of the nanoparticle-based facilitators was evaluated using BSA (1 mg/mL) as a comparative standard, as it is currently utilised as a facilitator by several laboratories and is commercially available in kits. The synthesized nanoparticles (AuNPs and BSA-coated AuNPs) were characterized by the presence of surface plasmon resonance (SPR) under UV-visible spectroscopy using a SYNERGY-HT multiwell plate reader (Bio-Tek, USA) using the Gen5 software in the range of 300–700 nm, at 1nm increments in the wavelength. Hydrodynamic size and zeta potential of the AuNPs and BSA-coated AuNPs were determined by transferring a 1% aqueous solution of nanoparticles into a disposable polystyrene cuvette and a standard zeta cuvette and measured using a Zetasizer Nano-ZS equipped with 4.0 Mw, 633 nm laser (Model ZEN3600, Malvern Instruments, Malvern, UK). The samples were analyzed three times at 25°C. The shape and size of the synthesised nanoparticles were imaged using Transmission Electron Microscopy (TEM) (Jeol JEM 1400 Plus microscope, USA), operating at voltages ranging from 20 to 120 kV. Infrared spectra of BSA-coated AuNPs were obtained using a PerkinElmer FT-IR Spectrometer. The aqueous solution of the samples was dried and mixed with potassium bromide (KBr) to obtain a fine powder pressed onto the discs. All spectra were measured at a resolution of 1/cm and over a wavelength range of 4000–400/cm. 4.2.5. nanoPCR-based amplification and STR Profiling: To understand the impact of melanin on the conventional DNA typing methods and evaluate the efficiency of nanoPCR in mitigating, controlling, , and facilitating (1, 2, and BSA) treated DNA samples were amplified using a GeneAmp PCR System 9700 Thermal Cycler. Seven conditions were included in the experimental protocol: a melanin-inhibited sample, an untreated control (DNA), control+BSA, control+AuNPs, control+BSA-coated AuNPs, and two mitigation groups with distinct facilitators: bare AuNPs (Facilitator 1) and BSA-coated AuNPs (Facilitator 2). To remove experimental bias and ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS standardise the reaction conditions, control groups containing BSA, AuNPs, and BSA-coated AuNPs were included. Additionally, BSA, a mitigating control group, has been examined to evaluate its relative effectiveness. The PCR reactions were performed using a half-reaction volume per the recommended protocol. The total reaction volume was kept at 10μl to ensure compatibility with the PCR system. For each reaction, 1μl of 2ng standard DNA served as a template, 1μl of inhibitor, and 2μls of facilitators 1, 2, and BSA were added where applicable. Final concentrations of the facilitators in the PCR mix were as follows: Bare AuNPs: 1.25 µg/mL (0.45nM); BSA- coated AuNPs: 0.954 nM (AuNPs functionalized with 18.9 ng of BSA, which corresponds to ~0.1 µg/mL BSA); Free BSA: 0.5 µg/mL. The cycling conditions are mentioned in Table 1 below. Table 1: Cycling conditions for amplification of control and treated DNA samples. Step Time Temperature Number of Cycles Initial Activation Step 2 min 96ºC ----- Three-step cycling Denaturation 10 sec 95ºC 27 Annealing 80 sec 59ºC Extension 10 min 60ºC The amplified products were stored at 4ºC for an hour. Post-PCR processing, capillary electrophoresis was performed on Applied Biosystems 3100/3500XL genetic analyzer using size standards provided with the kit where 1μl of the PCR product with 10 seconds of injection time was run. The obtained result was analyzed using GeneMapper ID-X v1.5 by Applied Biosystems, Foster City, CA. The peak detection threshold was set to 50 RFUs for the allele designation. Alleles were designated based on the number of alleles repeated with the help of an allelic ladder. All the samples were run in triplicate. 4.2.6. Assessment of DNA profile quality: To assure the quality of the DNA profile generated, the melanin and facilitator (1,2, and BSA) treated profiles were analysed for the reduced relative fluorescence intensities (RFU), allele dropout/drop-in, skewed peak height ratios, Total Peak Height (TPH), and global balance. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS TPH is the sum of the peak heights of both the heterozygous alleles and the height of the homozygous allele [58], Peak height of heterozygous alleles (Hhet) = H1+H2 Peak height of homozygous allele (Hhomo) = H-----(Taken as it is) Thus, TPH= Hhet + Hhomo Hhet and Hhomo represent the height of the heterozygous allele and the height of the homozygous allele, respectively. Global balance is shown as the total peak height of all the alleles in respective dye channels. The Peak Height Ratio (PHR) was calculated for heterozygous alleles, which is defined as the ratio of lower peak height (Heightl) to the higher peak height (Heighth) at the specific marker. Its value ranges between 0 and 1, where 0 indicates the condition where allele drop is observed, and 1 represents the ideal situation where both alleles have equal heights [58]. In this respect, the Mean local balance was also calculated, which is the mean of all the observed peak height ratios across the profile, where the value of the peak height ratio for a homozygous allele was kept as 1[59]. These results were compared among the different treatment groups to understand the impact of inhibitors and nano facilitators on the quality of the STR profile. Mean Local Balance = Average (Peak Height ratiohet + Peak Height ratiohomo) where, Peak Height ratiohet = (Heightl/Heighth)locus1 + (Heightl/Heighth)locus2 +….(Heightl/Heighth)locusN Peak Height ratiohomo = 1 for each homozygous loci. And, N is the number of heterozygous markers. Further, to assess the impact of facilitators on STR profile recovery and improvement, fold change was measured relative to the melanin-treated and control samples. Furthermore, TPH, PHR, MLB, and Global balance parameters were evaluated statistically using a non-parametric Kruskal-Wallis test at a 5 percent significance level, followed by Dunn's Multiple Comparison tests. Additionally, the efficacy of PCR facilitators was quantitatively assessed by statistically comparing BSA-coated gold nanoparticles with traditionally used BSA across various parameters. Because of the small sample sizes and inconsistent data distribution, all comparisons were examined using the Mann- Whitney U test, a non-parametric method appropriate for comparing two independent groups without assuming normality. The tests were two-tailed, and exact p-values were given as necessary. TPH and PHR values were compared in triplicate for each group, and medians and Hodges-Lehmann estimates were used to characterize the size of the discrepancies. With repeat ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS intensities, analyses were carried out in a same manner for dye channels using GraphPad Prism v8.0.2 (GraphPad Software, Boston, Massachusetts, USA; www.graphpad.com). A statistically significant result was defined as a p-value of less than 0.05. Data Availability Statement: I declare that the authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper. The molecular dynamics simulation movies used in this manuscript have been uploaded to the public data repository Zenodo to ensure transparency and accessibility, as per FAIR data principles. The Zenodo DOI is: https://doi.org/10.5281/zenodo.13933345. These movies are not part of any previously published or pending manuscripts. They serve as supporting data for the molecular interaction insights discussed in this submission and are being referenced here for the first time in a peer- reviewed publication. The data sharing on Zenodo is intended solely to comply with data availability standards and does not constitute prior or dual publication. References: 1. Mullis, K.B., The unusual origin of the polymerase chain reaction. Scientific American, 1990. 262(4): p. 56-65. 2. Galluzzi, L., et al., Real-time PCR applications for diagnosis of leishmaniasis. Parasites & vectors, 2018. 11: p. 1-13. 3. Matsuda, K., PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement. Advances in clinical chemistry, 2017. 80: p. 45-72. 4. Serrano-Cumplido, A., et al., Application of the PCR number of cycle threshold value (Ct) in COVID-19. Semergen, 2021. 47(5): p. 337-341. 5. Shahi, S., et al., Polymerase chain reaction (PCR)-based methods: promising molecular tools in dentistry. International journal of biological macromolecules, 2018. 117: p. 983- 992. 6. Mirmajlessi, S.M., et al., Real-time PCR applied to study on plant pathogens: potential applications in diagnosis-a review. Plant Protection Science, 2015. 51(4): p. 177-190. 7. Petralia, S. and S. Conoci, PCR technologies for point of care testing: progress and perspectives. ACS sensors, 2017. 2(7): p. 876-891. 8. McDonald, C., D. Taylor, and A. Linacre, PCR in Forensic Science: A Critical Review. Genes, 2024. 15(4): p. 438. 9. Weusten, J. and J. Herbergs, A stochastic model of the processes in PCR based amplification of STR DNA in forensic applications. Forensic Science International: Genetics, 2012. 6(1): p. 17-25. 10. Zhu, H., et al., PCR past, present and future. Biotechniques, 2020. 69(4): p. 317-325. 11. Singh, J., et al., A critical review on PCR, its types and applications. Int J Adv Res Biol Sci, 2014. 1(7): p. 65-80. 12. Vajpayee, K., et al., PCR Inhibitors and Facilitators-Their Role in Forensic DNA Analysis. Forensic Science International, 2023: p. 111773. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS 13. Herrling, T., K. Jung, and J. Fuchs, The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2008. 69(5): p. 1429-1435. 14. Guo, L., et al., Recent advances and progress on melanin: from source to application. International journal of molecular sciences, 2023. 24(5): p. 4360. 15. Bashkatov, A.N., et al., Estimate of the melanin content in human hairs by the inverse Monte-Carlo method using a system for digital image analysis. Quantum Electronics, 2006. 36(12): p. 1111. 16. Santos Nogueira, A.C. and I. Joekes, Hair color changes and protein damage caused by ultraviolet radiation. Journal of Photochemistry and Photobiology B: Biology, 2004. 74(2): p. 109-117. 17. Cordero, R.J. and A. Casadevall, Melanin. Current biology, 2020. 30(4): p. R142-R143. 18. Eckhart, L., et al., Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity. Biochem Biophys Res Commun, 2000. 271(3): p. 726-30. 19. Suenaga, E. and H. Nakamura, Evaluation of three methods for effective extraction of DNA from human hair. Journal of Chromatography B, 2005. 820(1): p. 137-141. 20. Yoshii, T., et al., [Water-soluble eumelanin as a PCR-inhibitor and a simple method for its removal]. Nihon Hoigaku Zasshi, 1993. 47(4): p. 323-9. 21. Thompson, R.E., G. Duncan, and B.R. McCord, An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification. J Forensic Sci, 2014. 59(6): p. 1517-29. 22. Vicente, A., et al., Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions. Histol Histopathol, 2019. 34(9): p. 1089-1096. 23. Yoshii, T., et al. PCR inhibitor: water-soluble melanin, which inhibits DNA polymerases and DNases. in Advances in Forensic Haemogenetics: 15th Congress of the International Society for Forensic Haemogenetics (Internationale Gesellschaft für forensische Hämogenetik eV), Venezia, 13–15 October 1993. 1994. Springer. 24. Alaeddini, R., Forensic implications of PCR inhibition—A review. Forensic Science International: Genetics, 2012. 6(3): p. 297-305. 25. Sarna, T., H.M. Swartz, and A. Zadlo, Interaction of melanin with metal ions modulates their cytotoxic potential. Applied Magnetic Resonance, 2022. 53(1): p. 105-121. 26. Solano, F., Melanins: skin pigments and much more—types, structural models, biological functions, and formation routes. New Journal of Science, 2014. 2014(1): p. 498276. 27. Vajpayee, K., D. Sagar, and H.R. Dash, Forensic DNA Typing: Inception, Methodology, and Technical Advancements. Forensic DNA Typing: Principles, Applications and Advancements, 2020: p. 3-26. 28. Wang, H., et al., A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples. Soil Biology and Biochemistry, 2017. 106: p. 109-118. 29. Kreader, C.A., Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Applied and environmental microbiology, 1996. 62(3): p. 1102-1106. 30. Farell, E.M. and G. Alexandre, Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates. BMC research notes, 2012. 5(1): p. 1-8. 31. Schriewer, A., A. Wehlmann, and S. Wuertz, Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8. Journal of Microbiological Methods, 2011. 85(1): p. 16-21. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS 32. Hamza, I.A. and M. Leifels, Assessment of PCR Inhibitor Removal Methods to Monitor Viruses in Environmental Water Samples: DAX-8 Outperforms Competitors. Water, Air, & Soil Pollution, 2023. 235(1): p. 20. 33. Zhang, Y., et al., Bovine thrombin enhances the efficiency and specificity of polymerase chain reaction. BioTechniques, 2014. 57(6): p. 289-294. 34. Strien, J., J. Sanft, and G. Mall, Enhancement of PCR amplification of moderate GC- containing and highly GC-rich DNA sequences. Molecular biotechnology, 2013. 54(3): p. 1048-1054. 35. Vajpayee, K., V. Paida, and R.K. Shukla, Nanoparticle-assisted PCR: fundamentals, mechanisms, and forensic implications. International Journal of Legal Medicine, 2025. 36. Tiwari, P.M., et al., Functionalized Gold Nanoparticles and Their Biomedical Applications. Nanomaterials, 2011. 1(1): p. 31-63. 37. Li, Y., et al., Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates. Protein Science, 1998. 7(5): p. 1116-1123. 38. Eom, S.H., J. Wang, and T.A. Steitz, Structure of Taq polymerase with DNA at the polymerase active site. Nature, 1996. 382(6588): p. 278-281. 39. Shi, X., et al., Spectroscopic investigation of the interactions between gold nanoparticles and bovine serum albumin. Chinese Science Bulletin, 2012. 57(10): p. 1109-1115. 40. Rossen, L., et al., Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol, 1992. 17(1): p. 37-45. 41. Schrader, C., et al., PCR inhibitors–occurrence, properties and removal. Journal of applied microbiology, 2012. 113(5): p. 1014-1026. 42. Sidstedt, M., et al., The impact of common PCR inhibitors on forensic MPS analysis. Forensic Science International: Genetics, 2019. 40: p. 182-191. 43. Bruce Mccord, Arianna Pionzio, and R. Thompson, Analysis of the Effect of a Variety of PCR Inhibitors on the Amplification of DNA Using Real Time PCR, Melt Curves and STR Analysis. The U.S. Department of Justice, Washington DC, Document No. 249148., 2014. 44. Roskoski, R., Modulation of Enzyme Activity, in xPharm: The Comprehensive Pharmacology Reference, S.J. Enna and D.B. Bylund, Editors. 2007, Elsevier: New York. p. 1-11. 45. Funes‐Huacca, M.E., et al., A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis. Electrophoresis, 2011. 32(9): p. 1084-1089. 46. Sidstedt, M., et al., Humic substances cause fluorescence inhibition in real-time polymerase chain reaction. Analytical biochemistry, 2015. 487: p. 30-37. 47. Dash, H.R., et al., Useful autosomal STR marker sets for forensic and paternity applications in the Central Indian population. Annals of Human Biology, 2021. 48(1): p. 37-48. 48. Kumar, A., et al., Genomic diversity at 22 STR loci (extended CODIS STR) in the population of Rajasthan, India. Gene Reports, 2021. 23: p. 101150. 49. Glock, B., et al., Additional variability at the D12S391 STR locus in an Austrian population sample: sequencing data and allele distribution. Forensic Science International, 1997. 90(3): p. 197-203. 50. Biedermann, A. and K.N. Kotsoglou, Forensic science and the principle of excluded middle: "Inconclusive" decisions and the structure of error rate studies. Forensic Sci Int Synerg, 2021. 3: p. 100147. 51. Giambernardi, T.A., U. Rodeck, and R.J. Klebe, Bovine serum albumin reverses inhibition of RT-PCR by melanin. Biotechniques, 1998. 25(4): p. 564-6. ACCEPTED MANUSCRIPT ARTICLE IN PRESS ARTICLE IN PRESS 52. Hu, Q., et al., A comparison of four methods for PCR inhibitor removal. Forensic science international: Genetics, 2015. 16: p. 94-97. 53. Silva Almeida Vicente, A.L., et al., Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions. 2019. 54. Nedumpully Govindan, P., L. Monticelli, and E. Salonen, Mechanism of taq DNA polymerase inhibition by fullerene derivatives: insight from computer simulations. The Journal of Physical Chemistry B, 2012. 116(35): p. 10676-10683. 55. Lu, C., et al., OPLS4: Improving Force Field Accuracy on Challenging Regimes of Chemical Space. Journal of Chemical Theory and Computation, 2021. 17(7): p. 4291-4300. 56. Janek, J. and J. Kolafa, Novel barostat implementation for molecular dynamics. The Journal of Chemical Physics, 2024. 160(18). 57. Kimling, J., et al., Turkevich method for gold nanoparticle synthesis revisited. The Journal of Physical Chemistry B, 2006. 110(32): p. 15700-15707. 58. Shrivastava, P., T. Jain, and R.K. Kumawat, Direct PCR amplification from saliva sample using non-direct multiplex STR kits for forensic DNA typing. Sci Rep, 2021. 11(1): p. 7112. 59. Hedman, J., R. Ansell, and A. Nordgaard, A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles. BMC Res Notes, 2010. 3: p. 290. ACCEPTED MANUSCRIPT ARTICLE IN PRESS