Mechanistic Insights into Melanin-Induced PCR Inhibition and Its NanoPCR-Based Mitigation

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Traditional mitigation measures, such as dilution or replacement of the polymerase, tend to result in loss of DNA and are not appropriate for low-template forensic samples. In this study, melanin-induced inhibition was explored by using molecular docking and dynamics simulation, which identified stable interaction with catalytic residues TYR671 and PHE667 (Kd = 31.76 ± 0.02 µM), interfering with polymerase function. Against this, gold nanoparticles (AuNPs) and BSA-coated AuNPs were employed as in situ facilitators. STR profiling established that BSA-coated AuNPs restored amplification efficiency significantly, with 2-fold improvement and recovery of heterozygous peaks. These results provide mechanistic insight into melanin inhibition and establish a nanotechnology-based approach to improve PCR results in recalcitrant forensic and clinical samples. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 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 many scientific domains, including genetics [2-5], molecular biology [5-7], and forensic research [8, 9], PCR could amplify small amounts of DNA into quantities acceptable 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 and contaminated samples [12]. These compounds interfere with the enzymatic action of the DNA polymerase, the integrity of the template, or 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]. In forensic caseworks, the samples derived from highly pigmented tissues, cremated remains, or degraded biological materials, melanin can induce significant PCR inhibition, leading to incomplete or total amplification failure [14]. This results in generating a partial or no STR profile, having allelic loss and reduction in peak height, which is a loss of its evidentiary value in a court of law [15]. 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 [14, 16] or the redox-active properties of melanin may generate reactive oxygen species (ROS), which cause oxidative damage to DNA and polymerase inhibition [17]. However, none of these mechanisms have been conclusively demonstrated, leaving a critical gap in DNA analysis. The physiochemical characteristics of melanin, such as hydrophobicity, metal-chelating activity, and redox activity, increase its strength as a PCR inhibitor [18]. Its 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 [19]. Structurally, melanin is classified into three broad types: eumelanin, pheomelanin, and neuromelanin. The most common type, eumelanin, produces black and brown pigmentation [20]. It mainly comprises 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) subunits, forming an insoluble and stable polymer [20]. Forensic evidence with melanin-rich biological tissues presents significant issues in DNA analysis. Hair, common biological evidence encountered in forensic casework, has high melanin content and can potentially be the source of inhibition-related mitochondrial and nuclear DNA analysis failures. Skin and epithelial cells commonly found in bloodstains, touch DNA, and sexual assault evidence contain variable levels of melanin that can potentially affect the efficiency of PCR. In cases involving severely charred bodies, decomposed human tissue, or soil-embedded evidence, melanin levels could be disproportionately elevated, adding extra intricacy to the DNA extraction and amplification protocol. Further, melanin-containing microbes and soil contaminants could add extra inhibitory compounds to DNA analysis [12, 21]. Given the importance of reliable DNA amplification in forensic analysis, elucidating and overcoming melanin-induced PCR inhibition is imperative. [22]. Even though efforts to overcome inhibition through dilution, the addition of BSA (bovine serum albumin), and the use of alternative chemical additives have been made, these approaches often compromise the sensitivity or result in incomplete DNA profiles. [12, 23]. The forensic community needs a more robust, potent, and targeted solution to overcome the failures of PCR caused by melanin. Addressing this issue will significantly improve the accuracy of forensic DNA analysis, especially in cases where biological samples that are heavily pigmented are the primary sources of evidence. In this regard, the present study seeks to explore melanin as a forensically significant PCR inhibitor, 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 refined protocols for forensic laboratories, thus ensuring improved success rates in DNA amplification of melanin-containing forensic samples, especially hair and other pigmented tissues. Results Molecular Docking and Molecular Dynamics Simulation Taq polymerase, a crucial component of the PCR, 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 S1 A). The polymerase domain, Klentaq domain, is subdivided further 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 polymerization. The Thumb has additional conserved residues ASN485, SER515, and LYS540, which have contact with the minor grooves of DNA. Additional conserved residues with contact with DNA are ARG573, GLU655, TYR671, ASN750, GLN745, HIS784, and ASP785 (Supplementary Figure S1 B). During amplification, Taq switches from the open conformation (1TAQ) to the closed, DNA-bound conformation (3KTQ) (Supplementary Figure S1 D) [ 24 , 25 ]. Based on the importance of the catalytic core, it was hypothesized that melanin could bind to the core and thereby suppress enzymatic activity. Molecular docking and dynamics simulations were employed to investigate this on 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—indicating direct interference with enzyme-template interaction (Supplementary Figure S3b). DNA deletion (3KTQΔDNA) decreased binding affinity to − 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. TYR671 positions DNA; PHE667 stabilizes nucleotide bases—both critical for polymerization (Supplementary Figure S3c). The docking score was enhanced in structure 3KTQΔDNAΔMg (–5.709 kcal/mol), which may be due to better accessibility to buried residues. 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), suggesting melanin can destabilize the enzyme in Mg²-depleted conditions by taking advantage of these binding pockets. 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 stabilizing residues (Fig. 1 ). In 1TAQ, RMSD varied from 1.5–10.5 Å, indicating instability. Initial interactions were dominated by ASP785 and HIS784; interaction by ARG573 was delayed. Transient contacts with ASN583 and HIS784 were observed (Figs. 1 B, 1 F), although instability indicated poor grip by melanin. 3KTQ displayed the lowest RMSD (2–5.4 Å), which illustrated high structural stability. Although melanin showed transient initial interactions with GLU507 (Fig. 1 C, 1 G), its binding was not stable. Still, the DNA-bound form of the enzyme appears to stabilize the overall architecture even if melanin's anchorage is poor. 3KTQΔDNA and 3KTQΔDNAΔMg had increased RMSD (6–16 Å and 1.5–10 Å, respectively), indicating destabilization. Stable hydrophobic contact with PHE667 and TYR671 in 3KTQΔDNA was maintained throughout the simulation (Figs. 1 D, 1 H). GLU615, PHE667, and TYR671 in 3KTQΔDNAΔMg regularly contacted melanin (Figs. 1 E, 1 I), consistent with the notion that loss of Mg²⁺ increases accessibility of melanin to functionally important residues. Combined, these findings establish a plausible inhibition mechanism: melanin targets conserved catalytic and structural residues (PHE667, TYR671, GLU615), destabilizing enzyme conformation and polymerase-DNA association. In particular, without stabilizing cofactors (DNA or Mg²⁺) available, hydrophobic and electrostatic interactions of melanin cause functional instability, which is responsible for its inhibitory effect in PCR. 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 (Fig. 2 ). This suggests that melanin is a moderate inhibitor, and a reversible interaction influences enzyme function through transient binding and structural modifications. Nanoparticle Characterization : Successful functionalization of AuNPs and BSA-coated AuNPs were verified using UV-visible spectroscopy (SPR band at ~ 520 nm redshifting 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 spectroscopy (presence of characteristic amide I and II bands at 1648 cm⁻¹ and 1549 cm⁻¹) (Fig. 3 ). nanoPCR-based STR genotyping : Melanin-treated samples had degraded STR profiles with no SE33 and Penta E amplification and partial dropout at D12S391. 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 221,704.7 RFU (range: 0–21,590.17 RFU). D12S391 and FGA had low PHRs (0.47 and 0.78), 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 (8,633 RFU), while blue (62,442.5 RFU), black (58,156.7 RFU), green (46,368.5 RFU), and red (46,104 RFU) had relatively higher intensities, suggesting differential quenching by melanin. Addition of Facilitator 1 (AuNPs) recovered the STR profile to some extent. Penta E and SE33 showed re-emergent peaks (4,196 RFU and ~ 229 RFU, respectively), and TPH was raised to 272,802.8 RFU (range: 229.3–24,377.8 RFU). Fluorescence was enhanced in all violet, blue, black, green, and red channels: 24,152.5 RFU, 57,263.8 RFU, 83,858.7 RFU, 49,743.7 RFU, and 57,784.2 RFU, respectively. Mean PHR was raised to 0.92, with FGA and D12S391 recovering to 0.96 and 0.8, respectively. Mean local balance was raised to 0.94. Though there was improvement in these, AuNPs were unable to abolish the inhibitory effect of melanin completely, though even loci-wise uniform improvement (absence of ski-slope) was seen. Facilitator 2 (BSA-coated AuNPs) improved further. Penta E (4,519 RFU) and SE33 (382, 384 RFU) had better recovery strength. TPH increased to 333,887.2 RFU (range: 383–21,777.5 RFU). Dye channel fluorescence improved to: blue (83,767.8 RFU), black (98,377.3 RFU), green (68,465.2 RFU), red (56,801.7 RFU), and violet (26,475.2 RFU). PHRs for FGA and D12S391 improved further to 0.79 and 0.97, respectively. Overall PHR averaged 0.93, with a mean local balance of 0.95, indicating uniform inhibition mitigation across loci. Fold-change analysis compared to melanin-only treatment showed 1.6-fold RFU increase in AuNPs and 2-fold increase in BSA-coated AuNPs, and the enhancement was highest at Penta E, D12S391, and SE33 (Fig. 4 ). Statistical comparison of total peak height (TPH) in control, melanin-inhibited, and nanoparticle-treated groups, by the Friedman test, indicated significant differences (Friedman statistic = 51.51, P < 0.0001). Pairwise comparisons then showed the melanin-inhibited samples to be statistically different from both the AuNP-treated (P = 0.0028) and Au:BSA-treated (P 0.9999), indicative of near-complete suppression of TPH levels. In targeted comparisons of the inhibitor and treated groups, gross elevation of TPH was found (Friedman statistic = 38.64, P < 0.0001), once more with Au:BSA optimal (P = 0.0462 and P < 0.0001, respectively). These findings validate the consistent performance of BSA-functionalized AuNPs in counteracting melanin inhibition in polymerase chain reaction (PCR) assays (Supplementary Table S3a, b). To identify allelic balance, PHRs among groups were contrasted. There were no statistically significant differences (Friedman statistic = 3.758, P = 0.2888), and pairwise tests were non-significant: control vs. inhibited (P > 0.9999), control vs. AuNPs (P = 0.2799), and control vs. Au:BSA (P > 0.9999). In the same manner, contrast between treated samples and melanin-only (Friedman statistic = 1.778, P = 0.4111) showed no differences. Thus, RFU was enhanced by AuNPs, but peak balance was not affected, and hence genotypic precision was maintained (Supplementary Table S3c, d). The signal dye analysis showed significant group differences (Friedman statistic = 11.64, P = 0.0018). While control and inhibited samples were statistically equivalent (P = 0.9816), Au:BSA enhanced fluorescence compared to the control group (P = 0.0044), thus confirming an enhancement in overall amplification. A direct comparison of inhibited and treated cohorts (Friedman statistic = 6.40, P = 0.0394) indicated that BSA-coated AuNPs alone statistically recovered the amplification (P = 0.0228). These findings indicate the superior rescue activity of BSA-functionalized nanoparticles with respect to local (TPH) and global (fluorescence) amplification measures (Supplementary Table S3e, f). The repeated benefits demonstrated across multiple measures confirm Au:BSA as an effective method for bypassing PCR inhibition in samples with degradation or inhibitors. Discussion The fundamental function 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 effective amplification, especially in low-template or degraded samples [ 12 ]. Of these, melanin has been reported time and again as a strong PCR inhibitor and is relatively high in pigmented clinical and forensic samples [ 21 ]. Nevertheless, there are clear descriptions of its inhibitory effects, but there has been insufficient knowledge on 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. The study shows that melanin is a mixed-mode, competitive, reversible, moderate-strength inhibitor of Taq polymerase that interacts non-covalently with the polymerase's catalytic residues and DNA binding groove, influencing the polymerase activity as well as template accessibility. Past experiments have either described the empirical trends of the inhibition or speculated intercalation of DNA as the principal pathway [ 12 , 21 , 26 – 28 ]. 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 [ 29 ]. Remarkably, the present study applies docking and MD analysis and identifies that melanin anchors on PHE667, TYR 671, and GLU 615, which are critical residues to support the polymerase structure, stabilize nucleotides, and elongate the strand. These interactions continued to persist in different conformations, particularly in the presence of Mg 2+ , or DNA-free conditions, implying the ability of melanin to unfavorably destabilize 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 [ 30 ]. Other previous findings based on STR are also supported by our data and even mechanistically elucidated. Allelic dropout, decrease in the rate of RFU, and imbalance between signals were jointly linked with melanin inhibition, as reported by Funes-Huacca et al. [ 31 ] and Sidstedt et al. [ 32 ], who also detected signal impairment in the melanin-contaminated forensic samples. 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 [ 33 , 34 ]. 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 [ 35 , 36 ]. More importantly, the study postulates that melanin acts through mixed-mode inhibition: it physically occludes DNA accessibility and also disrupts enzyme catalysis [ 29 ]. This further develops the classification of McCord and removes an uncertainty in the literature. The mechanism is of the dual-mode variety that accounts not only for the erratic performance of melanin across loci and channels of dyes but also why mitigation by dilution fails so often. To circumvent this issue, a nano-based mitigation approach using bare and BSA-functionalized gold nanoparticles was developed. As a previous study by Giambernardi et al. [ 37 ] demonstrated, the BSA was selected since serum albumin was observed to bind melanin with a high affinity via hydrophobic and π–π stacking interactions. Our Au:BSA NPs showed high recovery in TPH, fluorescence, as well as allelic balance, and these findings were statistically significant, proving the superiority of our BSA AuNPs, contrasting bare AuNPs. The outcomes of this can be compared to the conventional strategies like dilution, chelation, or enzyme replacement, which usually lead to template loss and incomplete recovery as reported by Hu et al. and Vicente et al [ 13 , 38 ]. This is an in situ clean-up method, in contrast to the conventional post-extraction clean-up methods that involve pre-treatment, and so DNA is lost. 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 the high signal rates in largely inhibited channels supporting the usefulness of Au:BSA in reclaiming the reaction efficiency without instating biased amplification kinetics. This work offers a conceptual breakthrough in inhibitor mitigation by combining molecular modeling with wet-lab 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 wider potential for low-quality samples, our dual-mode platform—which combines selective scavenging and enzyme protection—represents a matrix-compatible and mechanistically sound approach. In clinical studies, the strategy may be useful to diagnose melanomas, due to the confounding effect of high melanin content on DNA-based assays. This approach also provides an effective weapon to address weathered, decomposed, or pigmented samples in forensic genetics. On top of that, it is non-destructive and scalable, thus significantly broadening its use to cover archaeogenetics, wildlife forensics, and environmental DNA (sometimes requiring co-purifying inhibitors such as melanin, humic acids, and bile salts). The nanoparticle platform has opportunities to be expanded in the future 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. Online Methods 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 (HAuCl 4 ). Trisodium citrate (Na 3 C 6 H 5 O 7 ) (minimum assay 98%) and Bovine Serum Albumin (BSA) (minimum assay 98%) were obtained from Hi-media (Mumbai, India). 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 . Methods : Structure-based modelling and interaction analysis : 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 open and closed conformations of Taq polymerase (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 S1 A and C. 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. [ 39 ]. 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. [ 25 ] 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 in order to evaluate the stability and dynamic behavior 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 [ 40 ] using the Desmond module by Schrödinger (Schrödinger Release 2022-3: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2022, Maestro-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 + /Cl − ions to neutralize the system. The system was energy minimized 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 [ 41 ]. 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. 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 melanin binding. 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 triplicates. The dissociation constant (Kd), which is a measure of binding, was calculated using the standard binding equation $$\:\text{Y}=\frac{\text{B}\text{m}\text{a}\text{x}\times\:\left[\text{X}\right]}{\left[\text{X}\right]+\text{K}\text{d}}$$ 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. 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 study. Gold nanoparticles (AuNPs) were synthesized using the method elucidated by Enustun and Turkevich, where 1 mM of 45 mL HAuCl 4 .3H 2 O was reduced by using 38.8 mM of 5 mL freshly prepared trisodium citrate (TSC) [ 42 ]. The synthesized nanoparticles are citrated gold nanoparticles called 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 synthesized, where 1mg/ml BSA was incubated with 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 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 synthesized nanoparticles were imaged by Transmission Electron Microscopy (TEM) (Jeol JEM 1400 Plus microscope, USA) operating at voltages 20–120 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. nanoPCR-based amplification and STR Profiling To understand the impact of melanin on the conventional DNA typing method and evaluate the efficiency of nanoPCR in mitigation, control, melanin-inhibited, and facilitator (1 and 2) treated DNA samples were amplified using GeneAmp PCR System 9700 Thermal Cycler. 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 and 2 were added where applicable. 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 triplicates. Assessment of DNA profile quality To assure the quality of the DNA profile generated, the melanin and facilitator (1 and 2) treated profiles were analyzed for the reduced relative fluorescence intensities (RFU), allele dropout/drop-in, skewed peak height ratios, Total Peak Height (TPH), and global balance. TPH is the sum of the peak heights of both the heterozygous alleles and the height of the homozygous allele [ 43 ], Peak height of heterozygous alleles (H het ) = H 1 + H 2 Peak height of homozygous allele (H homo ) = H-----(Taken as it is) Thus, TPH = H het + H homo H het and H homo 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 (Height l ) to the higher peak height (Height h ) 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 [ 43 ]. 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[ 44 ]. 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 ratio het + Peak Height ratio homo ) where, Peak Height ratio het = (Height l /Height h ) locus1 + (Height l /Height h ) locus2 +….(Height l /Height h ) locusN Peak Height ratio homo = 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 Friedman test at a 5 percent significance level, followed by Dunn's Multiple Comparison tests using GraphPad Prism software (v8.0.2), GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com Declarations Supplementary Information : Supplementary Information is available for this paper. Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. 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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. 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. 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. Hu, Q., et al., A comparison of four methods for PCR inhibitor removal. Forensic science international: Genetics, 2015. 16 : p. 94-97. 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. 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. Janek, J. and J. Kolafa, Novel barostat implementation for molecular dynamics. The Journal of Chemical Physics, 2024. 160 (18). Kimling, J., et al., Turkevich method for gold nanoparticle synthesis revisited. The Journal of Physical Chemistry B, 2006. 110 (32): p. 15700-15707. 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. 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. Additional Declarations No competing interests reported. Supplementary Files SupplementaryfilemelaninFinal.docx Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Sep, 2025 Reviews received at journal 30 Aug, 2025 Reviewers agreed at journal 10 Aug, 2025 Reviews received at journal 09 Aug, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor invited by journal 25 Jul, 2025 Editor assigned by journal 18 Jul, 2025 Submission checks completed at journal 18 Jul, 2025 First submitted to journal 16 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7137528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":492847143,"identity":"cf3a8662-71b9-486c-8922-859c8b16f7ae","order_by":0,"name":"Kamayani Vajpayee","email":"","orcid":"","institution":"Ahmedabad University","correspondingAuthor":false,"prefix":"","firstName":"Kamayani","middleName":"","lastName":"Vajpayee","suffix":""},{"id":492847144,"identity":"a31bedec-9bb2-4378-9980-725d548f2018","order_by":1,"name":"Shriyansh Srivastava","email":"","orcid":"","institution":"Indian Institute of Technology Gandhinagar","correspondingAuthor":false,"prefix":"","firstName":"Shriyansh","middleName":"","lastName":"Srivastava","suffix":""},{"id":492847148,"identity":"64d01dfb-e8af-4036-9b57-92c38d592d10","order_by":2,"name":"Shivkant Sharma","email":"","orcid":"","institution":"Ingenomics Pvt. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Shivkant","middleName":"","lastName":"Sharma","suffix":""},{"id":492847155,"identity":"b948d729-6bf7-4927-b12c-7ba28badb585","order_by":3,"name":"Swadha Gupta","email":"","orcid":"","institution":"Central University of Gujarat","correspondingAuthor":false,"prefix":"","firstName":"Swadha","middleName":"","lastName":"Gupta","suffix":""},{"id":492847158,"identity":"c067182a-dde0-4e02-8d8a-aab17f1e85f0","order_by":4,"name":"Ashutosh Srivastava","email":"","orcid":"","institution":"Indian Institute of Technology Gandhinagar","correspondingAuthor":false,"prefix":"","firstName":"Ashutosh","middleName":"","lastName":"Srivastava","suffix":""},{"id":492847161,"identity":"97c33740-bd3e-4ed2-8ea6-6df9108956dd","order_by":5,"name":"Vidhi Paida","email":"","orcid":"","institution":"Ahmedabad University","correspondingAuthor":false,"prefix":"","firstName":"Vidhi","middleName":"","lastName":"Paida","suffix":""},{"id":492847163,"identity":"e427b63d-7124-4a84-905f-8a8750f3584f","order_by":6,"name":"Hirak Ranjan Dash","email":"","orcid":"","institution":"Centurion University of Technology and Management","correspondingAuthor":false,"prefix":"","firstName":"Hirak","middleName":"Ranjan","lastName":"Dash","suffix":""},{"id":492847166,"identity":"79cefbd8-e245-4b5c-b240-d83e9d7a920c","order_by":7,"name":"Anju Pappachan","email":"","orcid":"","institution":"Central University of Gujarat","correspondingAuthor":false,"prefix":"","firstName":"Anju","middleName":"","lastName":"Pappachan","suffix":""},{"id":492847168,"identity":"8185cc59-2f78-441c-acb2-ea4738aee71f","order_by":8,"name":"Ritesh K Shukla","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIie3Qv0oDMRzA8d8RuC6mXU9a3yFyQ4eK9yoJwt1Su7gIFswUJ3VVEJ/hpqJbjkBvyQNk7eLUoY4ZLOboH0SuwdEhX5LhuN8HfgQgFPqPIXcjDkAgjiRcAwUJzUGbX60E7Ymb1H8h8JNEYkPAMw/Du161sG+QDZOcSvt6Pumay8ptOGK8g2UbGSiEUqyBvT/lsrqfXVwdmwl1GxaMoy5tIwlCcb/Zh5iCSzxDrDRj4jZUjhyRA6RjrYCsIdXXy+2WrL0kBiwgKk0uFeZqS7iXpH0sElbqD6oG85o96yWRdF6k4hBxD/ZpxVlG6vx0sZzesId6nK5W09HJY0+3kp389U3djT3zoVAoFPL3DbvBZbICfrBSAAAAAElFTkSuQmCC","orcid":"","institution":"Ahmedabad University","correspondingAuthor":true,"prefix":"","firstName":"Ritesh","middleName":"K","lastName":"Shukla","suffix":""}],"badges":[],"createdAt":"2025-07-16 08:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7137528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7137528/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-35010-w","type":"published","date":"2026-01-22T15:58:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87944526,"identity":"625108dd-be53-4a6a-9a62-a27ebdfc79e0","added_by":"auto","created_at":"2025-07-30 15:56:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":629267,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 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) plot of molecular dynamics simulations showing the structural stability of melanin-bound protein complexes over 50ns. The RMSD values (in Å) were calculated for four different protein-melanin complexes for their initial structures. The protein models are color coded as: blue: 1TAQ, orange: 3KTQ, purple: 3KTQΔDNA, and brown: 3KTQΔDNAΔMg. (B-E) ligand-protein contact diagram shows the interaction profile of melanin with the different protein models during molecular dynamics simulation. The diagrams illustrate the frequency of the interactions between melanin and residues of the protein models across the simulation trajectory. 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. These plots provide insight into residues consistently involved in binding and their relative contributions to ligand stabilization. Specifically, F: 1TAQ, G: 3KTQ, H: 3KTQΔDNA, and I: 3KTQΔDNAΔMg.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7137528/v1/040574f2f5cf2b71b84c84ec.png"},{"id":87944529,"identity":"fecf15a1-5b18-4fcf-aa95-329e237a95ad","added_by":"auto","created_at":"2025-07-30 15:56:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345862,"visible":true,"origin":"","legend":"\u003cp\u003eTryptophan quenching analysis of protein in the presence of increasing melanin concentrations. A: Stern- Volmer plot showing the quenching efficiency of tryptophan residues, plotted as F\u003csub\u003e0\u003c/sub\u003e-F\u003csub\u003ec \u003c/sub\u003eversus melanin concentration (µM), where F\u003csub\u003e0 \u003c/sub\u003eis the fluorescence intensity of the protein in the absence of melanin and F\u003csub\u003ec \u003c/sub\u003eis the intensity in its presence.\u0026nbsp; B: Emission spectra of tryptophan fluorescence at increasing concentrations of melanin. A progressive decrease in fluorescence intensity with increasing melanin concentration indicates efficient quenching, suggestive of interactions with the tryptophan residues in the protein.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7137528/v1/0c3542b6582b9ea63c900db4.png"},{"id":87944528,"identity":"d65e8eee-1ff3-4ab0-aad3-ab15ce1716d9","added_by":"auto","created_at":"2025-07-30 15:56:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245655,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the synthesized bare AuNPs and Au: BSA. A) Representative TEM image taken from bare AuNPs (14 nm); B) Representative TEM image taken from Au:BSA NPs (26 nm); 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; 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.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7137528/v1/f02c7da9afc3d9a30d2f63f2.png"},{"id":87944527,"identity":"66990a8b-6606-4b27-8c4b-86074fdbba17","added_by":"auto","created_at":"2025-07-30 15:56:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":325182,"visible":true,"origin":"","legend":"\u003cp\u003eFold change analysis of 12 markers where reduced RFU and Allele dropout have been observed. Each graph represents the fold change following the treatment with the inhibitor melanin (black), and facilitators -AuNPS (Slate grey), and Au:BSA (grey), respectively, compared to the control samples.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7137528/v1/16672b2f164d35424e5c6dd8.png"},{"id":101151811,"identity":"41c6ce6a-720a-48f8-89d5-94f88bbc3a5b","added_by":"auto","created_at":"2026-01-26 16:05:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2501269,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7137528/v1/79108975-6826-4a73-8a23-2ca0680cb43c.pdf"},{"id":87944538,"identity":"63ece655-79c7-4eda-92a6-e66c8e429972","added_by":"auto","created_at":"2025-07-30 15:56:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2693609,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfilemelaninFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7137528/v1/09ae380c91d777e14a48bb3d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic Insights into Melanin-Induced PCR Inhibition and Its NanoPCR-Based Mitigation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe 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 many scientific domains, including genetics [2-5], molecular biology [5-7], and forensic research [8, 9], PCR could amplify small amounts of DNA into quantities acceptable 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 and contaminated samples [12]. These compounds interfere with the enzymatic action of the DNA polymerase, the integrity of the template, or reaction kinetics [12].\u003c/p\u003e\n\u003cp\u003eAmong the most potent and forensically relevant inhibitors, melanin is a polyphenolic biopolymer found in biological materials such as hair, skin, and decomposing tissue [13]. In forensic caseworks, the \u0026nbsp; samples derived from highly pigmented tissues, cremated remains, or degraded biological materials, melanin can induce significant PCR inhibition, leading to incomplete or total amplification failure [14]. This results in generating a partial or no STR profile, having allelic loss and reduction in peak height, which is a loss of its evidentiary value in a court of law [15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough 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 [14, 16] or the redox-active properties of melanin may generate reactive oxygen species (ROS), which cause oxidative damage to DNA and polymerase inhibition [17]. However, none of these mechanisms have been conclusively demonstrated, leaving a critical gap in DNA analysis.\u003c/p\u003e\n\u003cp\u003eThe physiochemical characteristics of melanin, such as hydrophobicity, metal-chelating activity, and redox activity, increase its strength as a PCR inhibitor [18]. Its structural characteristics promote non-specific protein interactions, divalent metal ion sequestration such as Mg\u0026sup2;⁺, and hindrance to DNA template binding, all of which can reduce the efficacy of DNA polymerase [19]. Structurally, melanin is classified into three broad types: eumelanin, pheomelanin, and neuromelanin. The most common type, eumelanin, produces black and brown pigmentation [20]. It mainly comprises 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) subunits, forming an insoluble and stable polymer [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eForensic evidence with melanin-rich biological tissues presents significant issues in DNA analysis. Hair, common biological evidence encountered in forensic casework, has high melanin content and can potentially be the source of inhibition-related mitochondrial and nuclear DNA analysis failures. Skin and epithelial cells commonly found in bloodstains, touch DNA, and sexual assault evidence contain variable levels of melanin that can potentially affect the efficiency of PCR. In cases involving severely charred bodies, decomposed human tissue, or soil-embedded evidence, melanin levels could be disproportionately elevated, adding extra intricacy to the DNA extraction and amplification protocol. Further, melanin-containing microbes and soil contaminants could add extra inhibitory compounds to DNA analysis [12, 21].\u003c/p\u003e\n\u003cp\u003eGiven the importance of reliable DNA amplification in forensic analysis, elucidating and overcoming melanin-induced PCR inhibition is imperative. [22]. Even though efforts to overcome inhibition through dilution, the addition of BSA (bovine serum albumin), and the use of alternative chemical additives have been made, these approaches often compromise the sensitivity or result in incomplete DNA profiles. [12, 23]. The forensic community needs a more robust, potent, and targeted solution to overcome the failures of PCR caused by melanin. Addressing this issue will significantly improve the accuracy of forensic DNA analysis, especially in cases where biological samples that are heavily pigmented are the primary sources of evidence.\u003c/p\u003e\n\u003cp\u003eIn this regard, the present study seeks to explore melanin as a forensically significant PCR inhibitor, 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\u0026apos;s effect but also guide the development of refined protocols for forensic laboratories, thus ensuring improved success rates in DNA amplification of melanin-containing forensic samples, especially hair and other pigmented tissues.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMolecular Docking and Molecular Dynamics Simulation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTaq polymerase, a crucial component of the PCR, 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 \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The polymerase domain, Klentaq domain, is subdivided further 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 polymerization. The Thumb has additional conserved residues ASN485, SER515, and LYS540, which have contact with the minor grooves of DNA. Additional conserved residues with contact with DNA are ARG573, GLU655, TYR671, ASN750, GLN745, HIS784, and ASP785 (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). During amplification, Taq switches from the open conformation (1TAQ) to the closed, DNA-bound conformation (3KTQ) (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Based on the importance of the catalytic core, it was hypothesized that melanin could bind to the core and thereby suppress enzymatic activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMolecular docking and dynamics simulations were employed to investigate this on 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—indicating direct interference with enzyme-template interaction (Supplementary Figure S3b). DNA deletion (3KTQΔDNA) decreased binding affinity to − 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. TYR671 positions DNA; PHE667 stabilizes nucleotide bases—both critical for polymerization (Supplementary Figure S3c). The docking score was enhanced in structure 3KTQΔDNAΔMg (–5.709 kcal/mol), which may be due to better accessibility to buried residues. 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), suggesting melanin can destabilize the enzyme in Mg²-depleted conditions by taking advantage of these binding pockets.\u003c/p\u003e\u003cp\u003eTo investigate complex stability in greater detail, 50-ns MD simulations were conducted. RMSD values monitored conformational drift, and protein-ligand (PL) contact analysis found stabilizing residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In 1TAQ, RMSD varied from 1.5–10.5 Å, indicating instability. Initial interactions were dominated by ASP785 and HIS784; interaction by ARG573 was delayed. Transient contacts with ASN583 and HIS784 were observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), although instability indicated poor grip by melanin. 3KTQ displayed the lowest RMSD (2–5.4 Å), which illustrated high structural stability. Although melanin showed transient initial interactions with GLU507 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), its binding was not stable. Still, the DNA-bound form of the enzyme appears to stabilize the overall architecture even if melanin's anchorage is poor. 3KTQΔDNA and 3KTQΔDNAΔMg had increased RMSD (6–16 Å and 1.5–10 Å, respectively), indicating destabilization. Stable hydrophobic contact with PHE667 and TYR671 in 3KTQΔDNA was maintained throughout the simulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). GLU615, PHE667, and TYR671 in 3KTQΔDNAΔMg regularly contacted melanin (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), consistent with the notion that loss of Mg²⁺ increases accessibility of melanin to functionally important residues. Combined, these findings establish a plausible inhibition mechanism: melanin targets conserved catalytic and structural residues (PHE667, TYR671, GLU615), destabilizing enzyme conformation and polymerase-DNA association. In particular, without stabilizing cofactors (DNA or Mg²⁺) available, hydrophobic and electrostatic interactions of melanin cause functional instability, which is responsible for its inhibitory effect in PCR.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTryptophan Quenching Assay\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe saturation binding experiment was performed using tryptophan fluorescence determination, and the Kd value for melanin was found to be 31.76 ± 0.02µM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that melanin is a moderate inhibitor, and a reversible interaction influences enzyme function through transient binding and structural modifications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eNanoparticle Characterization\u003c/b\u003e: Successful functionalization of AuNPs and BSA-coated AuNPs were verified using UV-visible spectroscopy (SPR band at ~ 520 nm redshifting 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 spectroscopy (presence of characteristic amide I and II bands at 1648 cm⁻¹ and 1549 cm⁻¹) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003enanoPCR-based STR genotyping\u003c/b\u003e: Melanin-treated samples had degraded STR profiles with no SE33 and Penta E amplification and partial dropout at D12S391. 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 221,704.7 RFU (range: 0–21,590.17 RFU). D12S391 and FGA had low PHRs (0.47 and 0.78), 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 (8,633 RFU), while blue (62,442.5 RFU), black (58,156.7 RFU), green (46,368.5 RFU), and red (46,104 RFU) had relatively higher intensities, suggesting differential quenching by melanin.\u003c/p\u003e\u003cp\u003eAddition of Facilitator 1 (AuNPs) recovered the STR profile to some extent. Penta E and SE33 showed re-emergent peaks (4,196 RFU and ~ 229 RFU, respectively), and TPH was raised to 272,802.8 RFU (range: 229.3–24,377.8 RFU). Fluorescence was enhanced in all violet, blue, black, green, and red channels: 24,152.5 RFU, 57,263.8 RFU, 83,858.7 RFU, 49,743.7 RFU, and 57,784.2 RFU, respectively. Mean PHR was raised to 0.92, with FGA and D12S391 recovering to 0.96 and 0.8, respectively. Mean local balance was raised to 0.94. Though there was improvement in these, AuNPs were unable to abolish the inhibitory effect of melanin completely, though even loci-wise uniform improvement (absence of ski-slope) was seen.\u003c/p\u003e\u003cp\u003eFacilitator 2 (BSA-coated AuNPs) improved further. Penta E (4,519 RFU) and SE33 (382, 384 RFU) had better recovery strength. TPH increased to 333,887.2 RFU (range: 383–21,777.5 RFU). Dye channel fluorescence improved to: blue (83,767.8 RFU), black (98,377.3 RFU), green (68,465.2 RFU), red (56,801.7 RFU), and violet (26,475.2 RFU). PHRs for FGA and D12S391 improved further to 0.79 and 0.97, respectively. Overall PHR averaged 0.93, with a mean local balance of 0.95, indicating uniform inhibition mitigation across loci.\u003c/p\u003e\u003cp\u003eFold-change analysis compared to melanin-only treatment showed 1.6-fold RFU increase in AuNPs and 2-fold increase in BSA-coated AuNPs, and the enhancement was highest at Penta E, D12S391, and SE33 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Statistical comparison of total peak height (TPH) in control, melanin-inhibited, and nanoparticle-treated groups, by the Friedman test, indicated significant differences (Friedman statistic = 51.51, P \u0026lt; 0.0001). Pairwise comparisons then showed the melanin-inhibited samples to be statistically different from both the AuNP-treated (P = 0.0028) and Au:BSA-treated (P \u0026lt; 0.0001) samples. No significant difference was observed, however, between the melanin-inhibited and control groups (P \u0026gt; 0.9999), indicative of near-complete suppression of TPH levels. In targeted comparisons of the inhibitor and treated groups, gross elevation of TPH was found (Friedman statistic = 38.64, P \u0026lt; 0.0001), once more with Au:BSA optimal (P = 0.0462 and P \u0026lt; 0.0001, respectively). These findings validate the consistent performance of BSA-functionalized AuNPs in counteracting melanin inhibition in polymerase chain reaction (PCR) assays (Supplementary Table S3a, b).\u003c/p\u003e\u003cp\u003eTo identify allelic balance, PHRs among groups were contrasted. There were no statistically significant differences (Friedman statistic = 3.758, P = 0.2888), and pairwise tests were non-significant: control vs. inhibited (P \u0026gt; 0.9999), control vs. AuNPs (P = 0.2799), and control vs. Au:BSA (P \u0026gt; 0.9999). In the same manner, contrast between treated samples and melanin-only (Friedman statistic = 1.778, P = 0.4111) showed no differences. Thus, RFU was enhanced by AuNPs, but peak balance was not affected, and hence genotypic precision was maintained (Supplementary Table S3c, d). The signal dye analysis showed significant group differences (Friedman statistic = 11.64, P = 0.0018). While control and inhibited samples were statistically equivalent (P = 0.9816), Au:BSA enhanced fluorescence compared to the control group (P = 0.0044), thus confirming an enhancement in overall amplification. A direct comparison of inhibited and treated cohorts (Friedman statistic = 6.40, P = 0.0394) indicated that BSA-coated AuNPs alone statistically recovered the amplification (P = 0.0228). These findings indicate the superior rescue activity of BSA-functionalized nanoparticles with respect to local (TPH) and global (fluorescence) amplification measures (Supplementary Table S3e, f). The repeated benefits demonstrated across multiple measures confirm Au:BSA as an effective method for bypassing PCR inhibition in samples with degradation or inhibitors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e The fundamental function 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 effective amplification, especially in low-template or degraded samples [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Of these, melanin has been reported time and again as a strong PCR inhibitor and is relatively high in pigmented clinical and forensic samples [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nevertheless, there are clear descriptions of its inhibitory effects, but there has been insufficient knowledge on 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. The study shows that melanin is a mixed-mode, competitive, reversible, moderate-strength inhibitor of Taq polymerase that interacts non-covalently with the polymerase's catalytic residues and DNA binding groove, influencing the polymerase activity as well as template accessibility. Past experiments have either described the empirical trends of the inhibition or speculated intercalation of DNA as the principal pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e–\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Remarkably, the present study applies docking and MD analysis and identifies that melanin anchors on PHE667, TYR 671, and GLU 615, which are critical residues to support the polymerase structure, stabilize nucleotides, and elongate the strand. These interactions continued to persist in different conformations, particularly in the presence of Mg\u003csup\u003e2+\u003c/sup\u003e, or DNA-free conditions, implying the ability of melanin to unfavorably destabilize 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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Other previous findings based on STR are also supported by our data and even mechanistically elucidated. Allelic dropout, decrease in the rate of RFU, and imbalance between signals were jointly linked with melanin inhibition, as reported by Funes-Huacca et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Sidstedt et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], who also detected signal impairment in the melanin-contaminated forensic samples. 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 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. More importantly, the study postulates that melanin acts through mixed-mode inhibition: it physically occludes DNA accessibility and also disrupts enzyme catalysis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This further develops the classification of McCord and removes an uncertainty in the literature. The mechanism is of the dual-mode variety that accounts not only for the erratic performance of melanin across loci and channels of dyes but also why mitigation by dilution fails so often. To circumvent this issue, a nano-based mitigation approach using bare and BSA-functionalized gold nanoparticles was developed. As a previous study by Giambernardi et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] demonstrated, the BSA was selected since serum albumin was observed to bind melanin with a high affinity via hydrophobic and π–π stacking interactions. Our Au:BSA NPs showed high recovery in TPH, fluorescence, as well as allelic balance, and these findings were statistically significant, proving the superiority of our BSA AuNPs, contrasting bare AuNPs. The outcomes of this can be compared to the conventional strategies like dilution, chelation, or enzyme replacement, which usually lead to template loss and incomplete recovery as reported by Hu et al. and Vicente et al [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This is an in situ clean-up method, in contrast to the conventional post-extraction clean-up methods that involve pre-treatment, and so DNA is lost. 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 the high signal rates in largely inhibited channels supporting the usefulness of Au:BSA in reclaiming the reaction efficiency without instating biased amplification kinetics. This work offers a conceptual breakthrough in inhibitor mitigation by combining molecular modeling with wet-lab 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 wider potential for low-quality samples, our dual-mode platform—which combines selective scavenging and enzyme protection—represents a matrix-compatible and mechanistically sound approach. In clinical studies, the strategy may be useful to diagnose melanomas, due to the confounding effect of high melanin content on DNA-based assays. This approach also provides an effective weapon to address weathered, decomposed, or pigmented samples in forensic genetics. On top of that, it is non-destructive and scalable, thus significantly broadening its use to cover archaeogenetics, wildlife forensics, and environmental DNA (sometimes requiring co-purifying inhibitors such as melanin, humic acids, and bile salts). The nanoparticle platform has opportunities to be expanded in the future 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.\u003c/p\u003e"},{"header":"Online Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eControl DNA (9948A; 2ng/\u0026micro;l) was procured from Ingenomics\u0026trade; Pvt Ltd, India, and was used to generate the control (inhibitor-free) DNA STR profiles.\u003c/p\u003e\n\u003cp\u003eTaq DNA polymerase (Cat. MB101-0500) was purchased from BioHelix Co. Ltd. (Taiwan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibitor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMelanin (inhibitor) and ammonium hydroxide (specific gravity 0.91) were purchased from Sigma Aldrich, US, and RFCL, Ltd, New Delhi, India, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanoparticle (Facilitator) Synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSigma Aldrich, USA, purchased chloroauric acid (HAuCl\u003csub\u003e4\u003c/sub\u003e). Trisodium citrate (Na\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) (minimum assay 98%) and Bovine Serum Albumin (BSA) (minimum assay 98%) were obtained from Hi-media (Mumbai, India). Milli-Q ultrapure water was used to make and reconstitute all the reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIngenomics\u0026trade; 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)\u003c/p\u003e\n\u003cp\u003eAll the statistical analyses were done using GraphPad Prism software (v8.0.2), GraphPad Software, Boston, Massachusetts, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructure-based modelling and interaction analysis\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCuration of crystal structure and small molecule data\u003c/strong\u003e: All the crystal structures corresponding to Taq polymerase (Uniprot: P19821) were retrieved from the Protein Data Bank (PDB). Two crystal structures corresponding to open and closed conformations of Taq polymerase (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 \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA and C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking and Molecular Dynamics Simulations\u003c/strong\u003e: Molecular Docking was carried out using the Glide module of Schr\u0026ouml;dinger (Schr\u0026ouml;dinger Release 2022-3: Maestro, Schr\u0026ouml;dinger, 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, Schr\u0026ouml;dinger, LLC, New York, NY, 2022; Impact, Schr\u0026ouml;dinger, LLC, New York, NY; Prime, Schr\u0026ouml;dinger, 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. [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. The 3KTQ protein structure was further prepared to generate three different crystal states: 3KTQ, 3KTQ without DNA (3KTQ\u0026Delta;DNA), and 3KTQ without DNA and Mg\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e ions (3KTQ\u0026Delta;DNA\u0026Delta;Mg). These four different models for Taq polymerase were used for docking studies.\u003c/p\u003e\n\u003cp\u003eThe Taq polymerase active site residues identified from a previous study by Eom et al. [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] were used for docking grid generation with the Receptor-Grid Generation module, ensuring accurate placement of melanin in the binding pocket (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Lastly, melanin was preprocessed using the Ligand Preparation module (LigPrep) (Schr\u0026ouml;dinger Release 2022-23: LigPrep, Schr\u0026ouml;dinger, LLC, New York, NY, 2023) and further docked to protein structure models (1TAQ, 3KTQ, 3KTQ\u0026Delta;DNA, 3KTQ\u0026Delta;DNA\u0026Delta;Mg) in extra precision mode (XP docking) using the Glide module (Schr\u0026ouml;dinger Release 2022-3: Glide, Schr\u0026ouml;dinger, LLC, New York, NY, 2022). The docking scores were used to evaluate the binding affinities of melanin to different Taq polymerase models.\u003c/p\u003e\n\u003cp\u003eMolecular dynamics (MD) simulations were performed on the docked complexes in order to evaluate the stability and dynamic behavior of the melanin\u0026ndash;Taq polymerase interactions observed through docking.\u003c/p\u003e\n\u003cp\u003eThe melanin-docked protein complexes were then used to perform molecular dynamics (MD) simulations. All the simulations were performed with an OPLS4 force field [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] using the Desmond module by Schr\u0026ouml;dinger (Schr\u0026ouml;dinger Release 2022-3: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2022, Maestro-Desmond Interoperability Tools, Schr\u0026ouml;dinger, 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\u003csup\u003e+\u003c/sup\u003e/Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions to neutralize the system. The system was energy minimized 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, Schr\u0026ouml;dinger, 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 Nos\u0026eacute;\u0026ndash;Hoover thermostat and Martyna\u0026ndash;Tobias\u0026ndash;Klein barostat [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. 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, Schr\u0026ouml;dinger, New York, NY, 2022.), with melanin selected as the ligand.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTryptophan Quenching Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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\u0026deg;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 melanin binding. 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\u0026ndash;20 aliquots of melanin from a 3.4mM stock solution were titrated against a fixed concentration of 0.1\u0026micro;M Taq polymerase. As melanin was introduced, changes in fluorescence were measured, and the possible binding was confirmed. The quenching assay was done in triplicates. The dissociation constant (Kd), which is a measure of binding, was calculated using the standard binding equation\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\text{Y}=\\frac{\\text{B}\\text{m}\\text{a}\\text{x}\\times\\:\\left[\\text{X}\\right]}{\\left[\\text{X}\\right]+\\text{K}\\text{d}}$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere Y represents the measured fluorescence signal, Bmax is the maximum binding capacity, [X] is the concentration of melanin, and Kd is the dissociation constant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibitor and Facilitator Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 1mg/ml stock solution of melanin was prepared in 0.5 N ammonium hydroxide, which was used as an inhibitor for the present study.\u003c/p\u003e\n\u003cp\u003eGold nanoparticles (AuNPs) were synthesized using the method elucidated by Enustun and Turkevich, where 1 mM of 45 mL HAuCl\u003csub\u003e4\u003c/sub\u003e.3H\u003csub\u003e2\u003c/sub\u003eO was reduced by using 38.8 mM of 5 mL freshly prepared trisodium citrate (TSC) [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. The synthesized nanoparticles are citrated gold nanoparticles called 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 synthesized, where 1mg/ml BSA was incubated with 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.\u003c/p\u003e\n\u003cp\u003eThe 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\u0026ndash;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\u0026deg;C. The shape and size of the synthesized nanoparticles were imaged by Transmission Electron Microscopy (TEM) (Jeol JEM 1400 Plus microscope, USA) operating at voltages 20\u0026ndash;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\u0026ndash;400/cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003enanoPCR-based amplification and STR Profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the impact of melanin on the conventional DNA typing method and evaluate the efficiency of nanoPCR in mitigation, control, melanin-inhibited, and facilitator (1 and 2) treated DNA samples were amplified using GeneAmp PCR System 9700 Thermal Cycler. The PCR reactions were performed using a half-reaction volume per the recommended protocol. The total reaction volume was kept at 10\u0026micro;l to ensure compatibility with the PCR system. For each reaction, 1\u0026micro;l of 2ng standard DNA served as a template, 1\u0026micro;l of inhibitor, and 2\u0026micro;ls of facilitators 1 and 2 were added where applicable. The cycling conditions are mentioned in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e below.\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCycling conditions for amplification of control and treated DNA samples.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStep\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of Cycles\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInitial Activation Step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96\u0026ordm;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eThree-step cycling\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDenaturation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 sec\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u0026ordm;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnnealing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80 sec\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59\u0026ordm;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExtension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u0026ordm;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe amplified products were stored at 4\u0026ordm;C 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 1\u0026micro;l 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 triplicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of DNA profile quality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assure the quality of the DNA profile generated, the melanin and facilitator (1 and 2) treated profiles were analyzed for the reduced relative fluorescence intensities (RFU), allele dropout/drop-in, skewed peak height ratios, Total Peak Height (TPH), and global balance.\u003c/p\u003e\n\u003cp\u003eTPH is the sum of the peak heights of both the heterozygous alleles and the height of the homozygous allele [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e],\u003c/p\u003e\n\u003cp\u003ePeak height of heterozygous alleles (H\u003csub\u003ehet\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;H\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003ePeak height of homozygous allele (H\u003csub\u003ehomo\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;H-----(Taken as it is)\u003c/p\u003e\n\u003cp\u003eThus, TPH\u0026thinsp;=\u0026thinsp;H\u003csub\u003ehet\u003c/sub\u003e + H\u003csub\u003ehomo\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003ehet\u003c/sub\u003e and H\u003csub\u003ehomo\u003c/sub\u003e represent the height of the heterozygous allele and the height of the homozygous allele, respectively.\u003c/p\u003e\n\u003cp\u003eGlobal 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 (Height\u003csub\u003el\u003c/sub\u003e) to the higher peak height (Height\u003csub\u003eh\u003c/sub\u003e) 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 [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. 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[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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.\u003c/p\u003e\n\u003cp\u003eMean Local Balance\u0026thinsp;=\u0026thinsp;Average (Peak Height ratio\u003csub\u003ehet\u003c/sub\u003e + Peak Height ratio\u003csub\u003ehomo\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003ewhere,\u003c/p\u003e\n\u003cp\u003ePeak Height ratio\u003csub\u003ehet =\u003c/sub\u003e (Height\u003csub\u003el\u003c/sub\u003e/Height\u003csub\u003eh\u003c/sub\u003e)\u003csub\u003elocus1\u003c/sub\u003e + (Height\u003csub\u003el\u003c/sub\u003e/Height\u003csub\u003eh\u003c/sub\u003e)\u003csub\u003elocus2\u003c/sub\u003e +\u0026hellip;.(Height\u003csub\u003el\u003c/sub\u003e/Height\u003csub\u003eh\u003c/sub\u003e)\u003csub\u003elocusN\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003ePeak Height ratio\u003csub\u003ehomo\u003c/sub\u003e = 1 for each homozygous loci.\u003c/p\u003e\n\u003cp\u003eAnd, N is the number of heterozygous markers.\u003c/p\u003e\n\u003cp\u003eFurther, 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 Friedman test at a 5 percent significance level, followed by Dunn\u0026apos;s Multiple Comparison tests using GraphPad Prism software (v8.0.2), GraphPad Software, Boston, Massachusetts, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e: Supplementary Information is available for this paper.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMullis, K.B., \u003cem\u003eThe unusual origin of the polymerase chain reaction.\u003c/em\u003e Scientific American, 1990. \u003cstrong\u003e262\u003c/strong\u003e(4): p. 56-65.\u003c/li\u003e\n\u003cli\u003eGalluzzi, L., et al., \u003cem\u003eReal-time PCR applications for diagnosis of leishmaniasis.\u003c/em\u003e Parasites \u0026amp; vectors, 2018. \u003cstrong\u003e11\u003c/strong\u003e: p. 1-13.\u003c/li\u003e\n\u003cli\u003eMatsuda, K., \u003cem\u003ePCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement.\u003c/em\u003e Advances in clinical chemistry, 2017. \u003cstrong\u003e80\u003c/strong\u003e: p. 45-72.\u003c/li\u003e\n\u003cli\u003eSerrano-Cumplido, A., et al., \u003cem\u003eApplication of the PCR number of cycle threshold value (Ct) in COVID-19.\u003c/em\u003e Semergen, 2021. \u003cstrong\u003e47\u003c/strong\u003e(5): p. 337-341.\u003c/li\u003e\n\u003cli\u003eShahi, S., et al., \u003cem\u003ePolymerase chain reaction (PCR)-based methods: promising molecular tools in dentistry.\u003c/em\u003e International journal of biological macromolecules, 2018. \u003cstrong\u003e117\u003c/strong\u003e: p. 983-992.\u003c/li\u003e\n\u003cli\u003eMirmajlessi, S.M., et al., \u003cem\u003eReal-time PCR applied to study on plant pathogens: potential applications in diagnosis-a review.\u003c/em\u003e Plant Protection Science, 2015. \u003cstrong\u003e51\u003c/strong\u003e(4): p. 177-190.\u003c/li\u003e\n\u003cli\u003ePetralia, S. and S. 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Kolafa, \u003cem\u003eNovel barostat implementation for molecular dynamics.\u003c/em\u003e The Journal of Chemical Physics, 2024. \u003cstrong\u003e160\u003c/strong\u003e(18).\u003c/li\u003e\n\u003cli\u003eKimling, J., et al., \u003cem\u003eTurkevich method for gold nanoparticle synthesis revisited.\u003c/em\u003e The Journal of Physical Chemistry B, 2006. \u003cstrong\u003e110\u003c/strong\u003e(32): p. 15700-15707.\u003c/li\u003e\n\u003cli\u003eShrivastava, P., T. Jain, and R.K. Kumawat, \u003cem\u003eDirect PCR amplification from saliva sample using non-direct multiplex STR kits for forensic DNA typing.\u003c/em\u003e Sci Rep, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 7112.\u003c/li\u003e\n\u003cli\u003eHedman, J., R. Ansell, and A. Nordgaard, \u003cem\u003eA ranking index for quality assessment of forensic DNA profiles forensic DNA profiles.\u003c/em\u003e BMC Res Notes, 2010. \u003cstrong\u003e3\u003c/strong\u003e: p. 290.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7137528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7137528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMelanin is a potent inhibitor of PCR that inhibits forensic DNA typing by binding Taq polymerase and interfering with its function, resulting in allele dropout and decreased peak heights. Traditional mitigation measures, such as dilution or replacement of the polymerase, tend to result in loss of DNA and are not appropriate for low-template forensic samples. In this study, melanin-induced inhibition was explored by using molecular docking and dynamics simulation, which identified stable interaction with catalytic residues TYR671 and PHE667 (Kd = 31.76 ± 0.02 µM), interfering with polymerase function. Against this, gold nanoparticles (AuNPs) and BSA-coated AuNPs were employed as in situ facilitators. STR profiling established that BSA-coated AuNPs restored amplification efficiency significantly, with 2-fold improvement and recovery of heterozygous peaks. These results provide mechanistic insight into melanin inhibition and establish a nanotechnology-based approach to improve PCR results in recalcitrant forensic and clinical samples.\u003c/p\u003e","manuscriptTitle":"Mechanistic Insights into Melanin-Induced PCR Inhibition and Its NanoPCR-Based Mitigation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 15:56:14","doi":"10.21203/rs.3.rs-7137528/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-12T13:53:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-30T09:16:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48950597762746916151280373122638391343","date":"2025-08-10T23:31:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-10T00:30:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244495614548012926838713164815951446119","date":"2025-07-29T15:59:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T06:14:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-25T15:27:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-19T03:56:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-18T09:48:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-16T08:15:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d4ed987-95b9-4d97-a0b1-1ac70d5953ca","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52333164,"name":"Biological sciences/Biochemistry"},{"id":52333165,"name":"Biological sciences/Biological techniques"},{"id":52333166,"name":"Biological sciences/Biotechnology"},{"id":52333167,"name":"Biological sciences/Genetics"},{"id":52333168,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-01-26T16:02:00+00:00","versionOfRecord":{"articleIdentity":"rs-7137528","link":"https://doi.org/10.1038/s41598-026-35010-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-22 15:58:42","publishedOnDateReadable":"January 22nd, 2026"},"versionCreatedAt":"2025-07-30 15:56:14","video":"","vorDoi":"10.1038/s41598-026-35010-w","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35010-w","workflowStages":[]},"version":"v1","identity":"rs-7137528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7137528","identity":"rs-7137528","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}