DNA Nanotechnology & Application Laboratory, CSIR-Institute of Minerals & Materials Technology, Bhubaneswar 751013, Odisha, India; Academy of Scientific & Innovative Research (AcSIR), Ghaziabad 201002, Uttar Pradesh, India.
School of Chemical Sciences, National Institute of Science Education and Research, Bhubaneswar 752050, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India.
Int J Biol Macromol. 2021 Apr 30;177:119-128. doi: 10.1016/j.ijbiomac.2021.02.095. Epub 2021 Feb 17.
Branched DNA (bDNA) nanostructures have emerged as self-assembled biomaterials and are being considered for biomedical applications. Herein, we report the biophysical interaction between self-assembled bDNA nanostructure with circulating protein bovine serum albumin (BSA) and cellular enzyme bovine liver catalase (BLC). The binding between bDNA and BSA or BLC was confirmed through the decrease in fluorescence spectra. The Stern-Volmer data supports for non-covalent bonding with ~1 binding site in case of BSA and BLC thus advocating a static binding. Furthermore, FTIR and ITC study confirmed the binding of bDNAs with proteins through hydrogen bonding and van der Waals interaction. The negative free energy observed in ITC represent spontaneous reaction for BLC-bDNA interaction. The biophysical interaction between bDNA nanostructures and proteins was also supported by DLS and zeta potential measurement. With an increase in bDNA concentrations up to 100 nM, no significant change in absorbance and CD spectra was observed for both BLC and BSA which suggests structural stability and unaffected secondary conformation of proteins in presence of bDNA. Furthermore, the catalytic activity of BLC was unaltered in presence of bDNAscr even with increasing the incubation period from 1 h to 24 h. Interestingly, the time-dependent decrease in activity of BLC was protected by bDNAmix. The thermal melting study suggests a higher Tm value for proteins in presence of bDNAmix which demonstrates that interaction with bDNAmix increases the thermal stability of proteins. Collectively these data suggest that self-assembled DNA nanostructure may bind to BSA for facilitating circulation in plasma or binding to intracellular proteins like BLC for stabilization, however the secondary conformation of protein or catalytic activity of enzyme is unaltered in presence of bDNA nanostructure. Thus, the newly established genomic sequence-driven self-assembled DNA nanostructure can be explored for in vitro or in vivo experimental work in recent future.
分支 DNA（bDNA）纳米结构已成为自组装生物材料，并被认为具有生物医学应用的潜力。本文报告了自组装 bDNA 纳米结构与循环蛋白牛血清白蛋白（BSA）和细胞酶牛肝过氧化氢酶（BLC）之间的生物物理相互作用。通过荧光光谱的降低，证实了 bDNA 与 BSA 或 BLC 之间的结合。Stern-Volmer 数据支持与 BSA 和 BLC 之间的非共价键合，约有 1 个结合位点，因此主张是静态结合。此外，FTIR 和 ITC 研究通过氢键和范德华相互作用证实了 bDNAs 与蛋白质的结合。ITC 中观察到的负自由能代表 BLC-bDNA 相互作用的自发反应。DLS 和 zeta 电位测量也支持 bDNA 纳米结构与蛋白质之间的生物物理相互作用。随着 bDNA 浓度增加到 100 nM，BSA 和 BLC 的吸收和 CD 光谱均未发生明显变化，这表明 bDNA 存在时蛋白质结构稳定且二级构象未受影响。此外，即使孵育时间从 1 h 延长至 24 h，bDNAscr 存在时 BLC 的催化活性也未改变。有趣的是，bDNAmix 的存在保护了 BLC 的活性随时间的下降。热融研究表明 bDNAmix 存在时蛋白质的 Tm 值更高，这表明与 bDNAmix 的相互作用增加了蛋白质的热稳定性。总的来说，这些数据表明，自组装 DNA 纳米结构可能与 BSA 结合以促进在血浆中的循环，或与细胞内蛋白质（如 BLC）结合以稳定，但 bDNA 纳米结构存在时蛋白质的二级构象或酶的催化活性保持不变。因此，新建立的基于基因组序列驱动的自组装 DNA 纳米结构可以在未来的体外或体内实验中进行探索。
