Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology.

Repurposing of genistein as anti-sickling agent: elucidation by multi spectroscopic, thermophoresis, and molecular modeling techniques.

Sickle cell disease (SCD) is a major medical problem in which mono-therapeutic interventions have so far shown only limited effectiveness. We studied the repurpose of genistein, which could prevent sickle hemoglobin from polymerizing under hypoxic conditions in this disease. Genistein an important nutraceutical molecule found in soybean. The present study examines the repurposing genistein as an anti- sickling agent. Genistein shows inhibition of Hb S polymerization as well as a sickle reversal. Also, we have explored the interaction of the genistein with sickle hemoglobin (Hb S), using fluorescence, far-UV-CD spectroscopy, MicroScale Thermophoresis (MST), FTIR, combined with molecular modeling computations. The quenching constant decreases with increasing temperature, a characteristic that coincides with the static type of quenching mechanism. Temperature-dependent fluorescence measurements and molecular modeling studies reveal that apart from the hydrogen bonding, electrostatic interactions also play a crucial role in genistein and Hb S complex formation. In silico, distribution prediction of adsorption, digestion, metabolism, excretion, and toxicity (ADME/Tox) based on physical and chemical properties show that genistein is nontoxic and has ideal drug properties. The helicity and thermophoretic mobility of Hb S was a change in the presence of genistein, which leads to the destabilizing the Hb S polymer was examined using CD and MST, respectively. Our results open up the possibility for a promising therapeutic approach for the SCD by repurposed genistein as an anti-sickling agent.Communicated by Ramaswamy H. Sarma.

Potential of isoquercitrin as antisickling agent: a multi-spectroscopic, thermophoresis and molecular modeling approach.

Sickle cell disease is an inherited disease caused by point mutation in hemoglobin (ß-globin gene). Under oxygen saturation, sickle hemoglobin form polymers, leading to rigid erythrocytes. The transition of the blood vessels is altered and initiated by the adhesion of erythrocytes, neutrophils and endothelial cells. Sickle Hemoglobin (HbS) polymerization is a major cause in red blood cells (RBC), promoting sickling and destruction of RBCs. Isoquercitrin, a medicinal bioactive compound found in various medicinal plants, has multiple health benefits. The present study examines the potential of isoquercitrin as an anti-sickle agent, showing a significant decrease in the rate of polymerization as well as sickling of RBCs. Isoquercitrin-induced graded alteration in absorbance and fluorescence of HbS, confirmed their interaction. A negative value of ΔG° strongly suggests that it is a spontaneous exothermic reaction induced by entropy. Negative ΔH° and positive ΔS° predicted that hydrogen and hydrophobic binding forces interfered with a hydrophobic microenvironment of ß6Val leading to polymerization inhibition of HbS. HbS-Isoquercitrin complex exhibits helical structural changes leading to destabilization of the HbS polymer as confirmed by CD spectroscopy. MST and DSC results indicate greater changes in thermophoretic mobility and thermal stability of sickle hemoglobin in the presence of isoquercitrin, respectively. These findings were also supported by molecular simulation studies using DOCK6 and GROMACS. Hence, we can conclude that isoquercitrin interacts with HbS through hydrogen bonding, which leads to polymerization inhibition. Consequently, isoquercitrin could potentially be used as a medication for the treatment of sickle cell disease.Communicated by Ramaswamy H. Sarma.

Alizarin interaction with sickle hemoglobin: elucidation of their anti-sickling properties by multi-spectroscopic and molecular modeling techniques.

Polymerization of hemoglobin S is a major cause of morbidity and mortality in sickle cell disease, which leads to sickling and destruction of red blood cell. Alizarin, a bioactive compound from Rubia cordifolia, is reported to be blood purifier. This study investigates the potential of alizarin as an anti-sickling agent, showing a significant decrease in the rate of polymerization, therefore inhibiting the rate of sickling with increasing concentration. Interaction studies indicated that the fluorescence intensity of sickle hemoglobin (Hb S) decreases gradually with increasing alizarin concentration. This suggests the static quenching, where binding constant and the number of binding sites were deduced at different temperatures. The negative values of Gibbs energy change (ΔG0) strongly suggest that it is entropy-driven spontaneous and exothermic reaction. Negative enthalpy (ΔH0) and positive entropy (ΔS0) stipulated that hydrogen and hydrophobic bonding forces were interfering in a hydrophobic micro-environment of ß6Val leading to Hb S polymerization inhibition. In circular dichroism (CD) spectra, Hb S in the presence of alizarin shows helical structural changes leading to destabilization of Hb S polymer. These findings were also supported by molecular docking simulation studies using DOCK6 and GROMACS. So, from these findings, we may conclude that alizarin interacts with Hb S through hydrogen bonding and leading to inhibition of Hb S polymerization. Consequently, alizarin may have potential use as an anti-sickle cell medication for sickle cell disorder. Communicated by Ramaswamy H. Sarma.