Water-resistant free-standing DNA-complexed films with antioxidant and H2O2-responsive activity.

Water-insoluble DNA complexes are suitable for producing free-standing DNA films due to their low water sensitivity, which prevents their rapid degradation in aqueous environments. Here, we proposed two types of free-standing films that exhibit low dissolution rates in water: low molecular weight chitosan (LCS)-DNA films and phosphatidylcholine (PC)-cetyltrimethylammonium (CTMA)-DNA films. The structure and binding characteristics of the LCS-DNA and PC-CTMA-DNA complexes were investigated with UV-Vis spectroscopy and via the fluorescent characteristics of daunorubicin bound to them. A simple drop-casting method was then adopted for both complexes to fabricate free-standing films. An increase in antioxidant activity and water-resistance of the LCS-DNA DNA film was observed when the molar ratio of LCS to DNA was increased, but the dissolution rate of the LCS-DNA film was also dependent on the ionic strength of the dissolving solution. Fourteen days were required to dissolve the LCS-DNA film in deionized water, whereas immediate dissolution was observed in 1× phosphate-buffered saline (PBS). Deformation of the PC-CTMA-DNA film was accelerated by H2O2, such that the PC-CTMA-DNA film was degraded after 21 days of immersion in 1× PBS with H2O2. Due to the low dissolution rate in water and antioxidant activity, the free-standing LCS-DNA film should be able to store and protect embedded clinical materials, such as proteins and intercalating drugs, from moisture and enable localized delivery of treatments to designated sites. Also, the free-standing PC-CTMA-DNA film could be a biocompatible candidate for use as a membrane or sensor for detecting the levels of reactive oxygen species.

DNA lattice growth with single, double, and triple double-crossover boundaries by stepwise self-assembly.

Construction of various nanostructures with nanometre-scale precision through various DNA building blocks depends upon self-assembly, base-pair complementarity and sequence programmability. During annealing, unit tiles are formed by the complementarity of base pairs in each strand. Enhancement of growth of target lattices is expected if seed lattices (i.e. boundaries for growth of target lattices) are initially present in a test tube during annealing. Although most processes for annealing DNA nanostructures adopt a one-step high temperature method, multi-step annealing provides certain advantages such as reusability of unit tiles and tuneability of lattice formation. We can construct target lattices effectively (through multi-step annealing) and efficiently (via boundaries) by multi-step annealing and combining boundaries. Here, we construct efficient boundaries made of single, double, and triple double-crossover DNA tiles for growth of DNA lattices. Two unit double-crossover DNA tile-based lattices and copy-logic implemented algorithmic lattices were introduced to test the growth of target lattices on boundaries. We used multi-step annealing to tune the formation of DNA crystals during fabrication of DNA crystals comprised of boundaries and target lattices. The formation of target DNA lattices was visualized using atomic force microscopy (AFM). The borders between boundaries and lattices in a single crystal were clearly differentiable from AFM images. Our method provides way to construct various types of lattices in a single crystal, which might generate various patterns and enhance the information capacity in a given crystal.

Ternary representation of N (N = 1 or 2)-input and 1-output algorithmic self-assembly demonstrated by DNA.

Deoxyribonucleic acid (DNA) is effective for molecular computation because of its high energy efficiency, high information density, and parallel-computing capability. Although logic implementation using DNA molecules is well established in binary systems (base value of 2) via decoration of hairpin structures on DNA duplexes, systems with base values of >2 (e.g. 3, corresponding to a ternary system) are rarely discussed owing to the complexity of the design and the experimental difficulties with DNA. In this study, DNA rule tiles that participate to form algorithmic DNA crystals exhibiting the ternary representation of an N (N = 1 or 2)-input and 1-output algorithmic assembly are conceived. The number of possible algorithmic patterns is [Formula: see text] in the ternary N-input and 1-output logic gate. Thus, the number of possible rules is 27 (=33) for a 1-input and 1-output algorithmic logic gate and 19 638 (=39) for a 2-input and 1-output algorithmic logic gate. Ternary bit information (i.e. 0-, 1-, and 2-bit) is encoded on rule tiles without hairpins and with short and long hairpins. We construct converged, line-like, alternating, and commutative patterns by implementing specific rules (TR00, TR05, TR07, and TR15, respectively) for the 1-input and 1-output gate and an ascending line-like pattern (with the rule of TR3785) for the 2-input and 1-output gate. Specific patterns generated on ternary-representing rule-embedded algorithmic DNA crystals are visualized via atomic force microscopy, and the errors during the growth of the crystals are analyzed (average error rates obtained for all experimental data are <4%). Our method can easily be extended to a system having base values of >3.

Band gap, dielectric constant, and susceptibility of DNA layers as controlled by vanadium ion concentration.

Deoxyribonucleic acid (DNA) doped with transition metal ions shows great versatility for molecular-based biosensors and bioelectronics. Methodologies for developing DNA lattices (formed by synthetic double-crossover tiles) and DNA layers (used by natural salmon) doped with vanadium ions (V3+), as well as an understanding of the physical characteristics of V3+-doped DNA nanostructures, are essential in practical applications in interdisciplinary research fields. Here, DNA lattices and layers doped with V3+ are constructed through substrate-assisted growth and drop-casting methods. In addition, enhanced physical characteristics such as the band gap energy, work function, dielectric constant, and susceptibility of V3+-doped DNA nanostructures with varying V3+ concentration ([V 3+ ]) are investigated. The critical concentration ([V 3+ ]C ) at a given amount of DNA was predicted based on an analysis of the phase transition of DNA lattices from crystalline to amorphous with specific [V 3+ ]. Generally, the [V 3+ ]C provided crucial information on the structural stability and extremum physical characteristics of V3+-doped DNA nanostructures due to the optimum incorporation of V3+ into DNA. We obtained the optical absorption spectra for energy band gap estimation; Raman spectra for identifying the preferential coordination sites of V3+ in DNA; x-ray photoelectron spectra to examine the chemical state, chemical composition, and functional groups; and ultraviolet photoelectron spectra to estimate the work function. In addition, we addressed the electrical properties (i.e. current, capacitance, dielectric constant, and storage energy) and magnetic properties (magnetic field-dependent and temperature-dependent magnetizations and susceptibility) of DNA layers in the presence of V3+. The development of biocompatible materials with specific optical, electrical, and magnetic properties is required for future applications because they must have designated functionality, high efficiency, and affordability.

The observation of resistive switching characteristics using transparent and biocompatible Cu2+-doped salmon DNA composite thin film.

For the potential switching bio-memory device application using DNA composite thin film, we fabricated and characterized the transparent and biocompatible resistive switching random access memory (RRAM) device within the structure stacking of Pt/Cu2+ doped salmon DNA/FTO, where Cu2+ doping into salmon DNA was solution-processed. The device shows good bipolar switching characteristics with SET and RESET processes at negative and positive sweeps, respectively, with switching memory window greater than 103 ratios. The device was observed to be in low resistance state as its pristine state and an initial RESET state was necessary to achieve programmable SET and RESET cycles. Based on the electrical characteristics of the Cu2+-doped salmon DNA-based RRAM device we propose a switching mechanism with the formation and rupture of conductive filaments due to the migration of Cu2+ during the electrical stress. Our understanding could contribute to the engineering of biomaterial memory switching medium for the environmentally benign, biocompatible and biodegradable memory storage devices.

Optoelectrical and mechanical properties of multiwall carbon nanotube-integrated DNA thin films.

Thin films made of deoxyribonucleic acid (DNA), dissolved in an aqueous solution, and cetyltrimethyl-ammonium-modified DNA (CDNA), dissolved in an organic solvent, utilising multiwall carbon nanotubes (MWCNTs) are not yet well-understood for use in optoelectronic device and sensor applications. In this study, we fabricate MWCNT-integrated DNA and CDNA thin films using the drop-casting method. We also characterise the optical properties (i.e. absorption spectra, Fourier-transform infrared spectra, Raman spectra, photoluminescence, and time-of-flight secondary ion mass spectrometry) to study spectral absorption, interaction, functional group, chirality, and compositional moiety and its distribution of MWCNTs in DNA and CDNA thin films. The electrical property for conductance and the mechanical characterisations of hardness, modulus and elasticity for stability are also discussed. Lastly, to show the feasibility of directional alignment of MWCNTs in DNA thin films, we perform an alignment experiment with MWCNTs in DNA via brushing and shearing methods, and we evaluate the results using polarised optical microscopy. Our simple methodology to align ingredients in DNA and CDNA thin films leveraging various optical, electrical and mechanical properties, provides great potential for the development of efficient devices and sensors.

Molecular Viscosity Sensors with Two Rotators for Optimizing the Fluorescence Intensity-Contrast Trade-Off.

A series of fluorescent molecular rotors obtained by introducing two rotational groups ("rotators"), which exhibit different rotational and electron-donating abilities, are discussed. Whereas the control molecular rotor, PH, includes a single rotator (the widely used phenyl group), the PO molecular rotors consist of two rotators (a phenyl group and an alkoxy group), which exhibit simultaneous strongly electron-donating and easy rotational abilities. Compared with the control rotor PH, PO molecular rotors exhibited one order of magnitude higher quantum yield (fluorescence intensity) and simultaneously exhibited significantly higher fluorescence contrast. These properties are directly related to the strong electron-donating ability and low energy barrier of rotation of the alkoxy group, as confirmed by dynamic fluorescence experiments and quantum chemical calculations. The PO molecular rotors exhibited two fluorescence relaxation pathways, whereas the PH molecular rotor exhibited a single fluorescence relaxation pathway. Cellular fluorescence imaging with PO molecular rotors for mapping cellular viscosity was successfully demonstrated.

Magneto-optical and thermal characteristics of magnetite nanoparticle-embedded DNA and CTMA-DNA thin films.

Recently, DNA molecules embedded with magnetite (Fe3O4) nanoparticles (MNPs) drew much attention for their wide range of potential usage. With specific intrinsic properties such as low optical loss, high transparency, large band gap, high dielectric constant, potential for molecular recognition, and their biodegradable nature, the DNA molecule can serve as an effective template or scaffold for various functionalized nanomaterials. With the aid of cetyltrimethylammonium (CTMA) surfactant, DNA can be used in organic-based applications as well as water-based ones. Here, DNA and CTMA-DNA thin films with various concentrations of MNPs fabricated by the drop-casting method have been characterized by optical absorption, refractive index, Raman, and cathodoluminescence measurements to understand the binding, dispersion, chemical identification/functional modes, and energy transfer mechanisms, respectively. In addition, magnetization was measured as a function of either applied magnetic field or temperature in field cooling and zero field cooling. Saturation magnetization and blocking temperature demonstrate the importance of MNPs in DNA and CTMA-DNA thin films. Finally, we examine the thermal stabilities of MNP-embedded DNA and CTMA-DNA thin films through thermogravimetric analysis, derivative thermogravimetry, and differential thermal analysis. The unique optical, magnetic, and thermal characteristics of MNP-embedded DNA and CTMA-DNA thin films will prove important to fields such as spintronics, biomedicine, and function-embedded sensors and devices.

Fabrication and Characterization of Finite-Size DNA 2D Ring and 3D Buckyball Structures.

In order to incorporate functionalization into synthesized DNA nanostructures, enhance their production yield, and utilize them in various applications, it is necessary to study their physical stabilities and dynamic characteristics. Although simulation-based analysis used for DNA nanostructures provides important clues to explain their self-assembly mechanism, structural function, and intrinsic dynamic characteristics, few studies have focused on the simulation of DNA supramolecular structures due to the structural complexity and high computational cost. Here, we demonstrated the feasibility of using normal mode analysis for relatively complex DNA structures with larger molecular weights, i.e., finite-size DNA 2D rings and 3D buckyball structures. The normal mode analysis was carried out using the mass-weighted chemical elastic network model (MWCENM) and the symmetry-constrained elastic network model (SCENM), both of which are precise and efficient modeling methodologies. MWCENM considers both the weight of the nucleotides and the chemical bonds between atoms, and SCENM can obtain mode shapes of a whole structure by using only a repeated unit and its connectivity with neighboring units. Our results show the intrinsic vibrational features of DNA ring structures, which experience inner/outer circle and bridge motions, as well as DNA buckyball structures having overall breathing and local breathing motions. These could be used as the fundamental basis for designing and constructing more complicated DNA nanostructures.

Charge Transport in 2D DNA Tunnel Junction Diodes.

Recently, deoxyribonucleic acid (DNA) is studied for electronics due to its intrinsic benefits such as its natural plenitude, biodegradability, biofunctionality, and low-cost. However, its applications are limited to passive components because of inherent insulating properties. In this report, a metal-insulator-metal tunnel diode with Au/DNA/NiOx junctions is presented. Through the self-aligning process of DNA molecules, a 2D DNA nanosheet is synthesized and used as a tunneling barrier, and semitransparent conducting oxide (NiOx ) is applied as a top electrode for resolving metal penetration issues. This molecular device successfully operates as a nonresonant tunneling diode, and temperature-variable current-voltage analysis proves that Fowler-Nordheim tunneling is a dominant conduction mechanism at the junctions. DNA-based tunneling devices appear to be promising prototypes for nanoelectronics using biomolecules.

Electromagnetic and optical characteristics of Nb5+-doped double-crossover and salmon DNA thin films.

We report the fabrication and physical characteristics of niobium ion (Nb5+)-doped double-crossover DNA (DX-DNA) and salmon DNA (SDNA) thin films. Different concentrations of Nb5+ ([Nb5+]) are coordinated into the DNA molecules, and the thin films are fabricated via substrate-assisted growth (DX-DNA) and drop-casting (SDNA) on oxygen plasma treated substrates. We conducted atomic force microscopy to estimate the optimum concentration of Nb5+ ([Nb5+]O = 0.08 mM) in Nb5+-doped DX-DNA thin films, up to which the DX-DNA lattices maintain their structures without deformation. X-ray photoelectron spectroscopy (XPS) was performed to probe the chemical nature of the intercalated Nb5+ in the SDNA thin films. The change in peak intensities and the shift in binding energy were witnessed in XPS spectra to explicate the binding and charge transfer mechanisms between Nb5+ and SDNA molecules. UV-visible, Raman, and photoluminescence (PL) spectra were measured to determine the optical properties and thus investigate the binding modes, Nb5+ coordination sites in Nb5+-doped SDNA thin films, and energy transfer mechanisms, respectively. As [Nb5+] increases, the absorbance peak intensities monotonically increase until ∼[Nb5+]O and then decrease. However, from the Raman measurements, the peak intensities gradually decrease with an increase in [Nb5+] to reveal the binding mechanism and binding sites of metal ions in the SDNA molecules. From the PL, we observe the emission intensities to reduce them at up to ∼[Nb5+]O and then increase after that, expecting the energy transfer between the Nb5+ and SDNA molecules. The current-voltage measurement shows a significant increase in the current observed as [Nb5+] increases in the SDNA thin films when compared to that of pristine SDNA thin films. Finally, we investigate the temperature dependent magnetization in which the Nb5+-doped SDNA thin films reveal weak ferromagnetism due to the existence of tiny magnetic dipoles in the Nb5+-doped SDNA complex.

Phase, current, absorbance, and photoluminescence of double and triple metal ion-doped synthetic and salmon DNA thin films.

We fabricated synthetic double-crossover (DX) DNA lattices and natural salmon DNA (SDNA) thin films, doped with 3 combinations of double divalent metal ions (M2+)-doped groups (Co2+-Ni2+, Cu2+-Co2+, and Cu2+-Ni2+) and single combination of a triple M2+-doped group (Cu2+-Ni2+-Co2+) at various concentrations of M2+ ([M2+]). We evaluated the optimum concentration of M2+ ([M2+]O) (the phase of M2+-doped DX DNA lattices changed from crystalline (up to ([M2+]O) to amorphous (above [M2+]O)) and measured the current, absorbance, and photoluminescent characteristics of multiple M2+-doped SDNA thin films. Phase transitions (visualized in phase diagrams theoretically as well as experimentally) from crystalline to amorphous for double (Co2+-Ni2+, Cu2+-Co2+, and Cu2+-Ni2+) and triple (Cu2+-Ni2+-Co2+) dopings occurred between 0.8 mM and 1.0 mM of Ni2+ at a fixed 0.5 mM of Co2+, between 0.6 mM and 0.8 mM of Co2+ at a fixed 3.0 mM of Cu2+, between 0.6 mM and 0.8 mM of Ni2+ at a fixed 3.0 mM of Cu2+, and between 0.6 mM and 0.8 mM of Co2+ at fixed 2.0 mM of Cu2+ and 0.8 mM of Ni2+, respectively. The overall behavior of the current and photoluminescence showed increments as increasing [M2+] up to [M2+]O, then decrements with further increasing [M2+]. On the other hand, absorbance at 260 nm showed the opposite behavior. Multiple M2+-doped DNA thin films can be used in specific devices and sensors with enhanced optoelectric characteristics and tunable multi-functionalities.

Growth and Detachment of 5 Helix DNA Ribbons.

We report on the concentration-dependent surface-assisted growth and time-temperature-dependent detachment of one-dimensional 5 helix DNA ribbons (5HR) on a mica substrate. The growth coverage ratio was determined by varying the concentration of the 5HR strands in a test tube, and the detachment rate of 5HR on mica was determined by varying the incubation time at a fixed temperature on a heat block. The topological changes in the concentration-dependent attachment and the time-temperature-dependent detachment for 5HR on mica were observed via atomic force microscopy. The observations indicate that 5HR started to grow on mica at ~10 nM and provided full coverage at ~50 nM. In contrast, 5HR at 65 °C started to detach from mica after 5 min and was completely removed after 10 min. The growth and detachment coverage show a sinusoidal variation in the growth ratio and a linear variation with a rate of detachment of 20%/min, respectively. The physical parameters that control the stability of the DNA structures on a given substrate should be studied to successfully integrate DNA structures for physical and chemical applications.

Broadband supercontinuum generation using a hollow optical fiber filled with copper-ion-modified DNA.

We experimentally demonstrated supercontinuum generation through a hollow core photonic bandgap fiber (HC-PBGF) filled with DNA nanocrystals modified by copper ions in a solution. Both double-crossover nano DNA structure and copper-ion-modified structure provided a sufficiently high optical nonlinearity within a short length of hollow optical fiber. Adding a higher concentration of copper ion into the DNA nanocrystals, the bandwidth of supercontinuum output was monotonically increased. Finally, we achieved the bandwidth expansion of about 1000 nm to be sufficient for broadband multi-spectrum applications.

Construction and characterization of Cu²âº, Ni²âº, Zn²âº, and Co²âº modified-DNA crystals.

We studied the physical characteristics of modified-DNA (M-DNA) double crossover crystals fabricated via substrate-assisted growth with various concentrations of four different divalent metallic ions, Cu(2+), Ni(2+), Zn(2+), and Co(2+). Atomic force microscopy (AFM) was used to test the stability of the M-DNA crystals with different metal ion concentrations. The AFM images show that M-DNA crystals formed without deformation at up to the critical concentrations of 6 mM of [Cu(2+)], 1.5 mM of [Ni(2+)], 1 mM of [Zn(2+)], and 1 mM of [Co(2+)]. Above these critical concentrations, the M-DNA crystals exhibited deformed, amorphous structures. Raman spectroscopy was then used to identify the preference of the metal ion coordinate sites. The intensities of the Raman bands gradually decreased as the concentration of the metal ions increased, and when the metal ion concentrations increased beyond the critical values, the Raman band of the amorphous M-DNA was significantly suppressed. The metal ions had a preferential binding order in the DNA molecules with G-C and A-T base pairs followed by the phosphate backbone. A two-probe station was used to measure the electrical current-voltage properties of the crystals which indicated that the maximum currents of the M-DNA complexes could be achieved at around the critical concentration of each ion. We expect that the functionalized ion-doped M-DNA crystals will allow for efficient devices and sensors to be fabricated in the near future.

Long dsRNA-mediated RNA interference and immunostimulation: a targeted delivery approach using polyethyleneimine based nano-carriers.

RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

Ternary and senary representations using DNA double-crossover tiles.

The information capacity of DNA double-crossover (DX) tiles was successfully increased beyond a binary representation to higher base representations. By controlling the length and the position of DNA hairpins on the DX tile, ternary and senary (base-3 and base-6) digit representations were realized and verified by atomic force microscopy. Also, normal mode analysis was carried out to study the mechanical characteristics of each structure.

Observation of Ultrahigh Photoconductivity in DNA-MoS2 Nano-Biocomposite.

A nano-biocomposite film with ultrahigh photoconductivity remains elusive and critical for bio-optoelectronic applications. A uniform, well-connected, high-concentration nanomaterial network in the biological matrix remains challenging to achieve high photoconductivity. Wafer-scale continuous nano-biocomposite film without surface deformations and cracks plays another major obstacle. Here ultrahigh photoconductivity is observed in deoxyribonucleic acid-molybdenum disulfide (DNA-MoS2) nano-biocomposite film by incorporating a high-concentration, well-percolated, and uniform MoS2 network in the ss-DNA matrix. This is achieved by utilizing DNA-MoS2 hydrogel formation, which results in crack-free, wafer-scale DNA-MoS2 nano-biocomposite films. Ultra-high photocurrent (5.5 mA at 1 V) with a record-high on/off ratio (1.3 × 106) is observed, five orders of magnitude higher than conventional biomaterials (≈101) reported so far. The incorporation of the Wely semimetal (Bismuth) as an electrical contact exhibits ultrahigh photoresponsivity (2.6 × 105 A W-1). Such high photoconductivity in DNA-MoS2 nano-biocomposite could bridge the gap between biology, electronics, and optics for innovative biomedicine, bioengineering, and neuroscience applications.

Information Density Enhancement Using Lossy Compression in DNA Data Storage.

This study develops two deoxyribonucleic acid (DNA) lossy compression models, Models A and B, to encode grayscale images into DNA sequences, enhance information density, and enable high-fidelity image recovery. These models, distinguished by their handling of pixel domains and interpolation methods, offer a novel approach to data storage for DNA. Model A processes pixels in overlapped domains using linear interpolation (LI), whereas Model B uses non-overlapped domains with nearest-neighbor interpolation (NNI). Through a comparative analysis with Joint Photographic Experts Group (JPEG) compression, the DNA lossy compression models demonstrate competitive advantages in terms of information density and image quality restoration. The application of these models to the Modified National Institute of Standards and Technology (MNIST) dataset reveals their efficiency and the recognizability of decompressed images, which is validated by convolutional neural network (CNN) performance. In particular, Model B2, a version of Model B, emerges as an effective method for balancing high information density (surpassing over 20 times the typical densities of two bits per nucleotide) with reasonably good image quality. These findings highlight the potential of DNA-based data storage systems for high-density and efficient compression, indicating a promising future for biological data storage solutions.

Comparison of Statin With Ezetimibe Combination Therapy Versus Statin Monotherapy for Primary Prevention in Middle-Aged Adults.

BACKGROUND AND OBJECTIVES: Lipid lowering therapy is essential to reduce the risk of major cardiovascular events; however, limited evidence exists regarding the use of statin with ezetimibe as primary prevention strategy for middle-aged adults. We aimed to investigate the impact of single pill combination therapy on clinical outcomes in relatively healthy middle-aged patients when compared with statin monotherapy. METHODS: Using the Korean National Health Insurance Service database, a propensity score match analysis was performed for baseline characteristics of 92,156 patients categorized into combination therapy (n=46,078) and statin monotherapy (n=46,078) groups. Primary outcome was composite outcomes, including death, coronary artery disease, and ischemic stroke. And secondary outcome was all-cause death. The mean follow-up duration was 2.9±0.3 years. RESULTS: The 3-year composite outcomes of all-cause death, coronary artery disease, and ischemic stroke demonstrated no significant difference between the 2 groups (10.3% vs. 10.1%; hazard ratio (HR), 1.022; 95% confidence interval [CI], 0.980-1.064; p=0.309). Meanwhile, the 3-year all-cause death rate was lower in the combination therapy group than in the statin monotherapy group (0.2% vs. 0.4%; p<0.001), with a significant HR of 0.595 (95% CI, 0.460-0.769; p<0.001). Single pill combination therapy exhibited consistently lower mortality rates across various subgroups. CONCLUSIONS: Compared to the statin monotherapy, the combination therapy for primary prevention showed no difference in composite outcomes but may reduce mortality risk in relatively healthy middle-aged patients. However, since the study was observational, further randomized clinical trials are needed to confirm these findings.