Yes / Polymerase chain reaction (PCR) has become a powerful tool for detecting various diseases due to its high sensitivity and specificity. However, the long thermocycling time and the bulky system have limited the application of PCR devices in Point-of-care testing. Herein, we have proposed an efficient, low-cost, and hand-hold PCR microdevice, mainly including a control module based on water-cooling technology and an amplification module fabricated by 3D printing. The whole device is tiny and can be easily hand-held with a size of about 110 mm × 100 mm × 40 mm and a weight of about 300 g at a low cost of about $170.83. Based on the water-cooling technology, the device can efficiently perform 30 thermal cycles within 46 min at a heating/cooling rate of 4.0/8.1 ℃/s. To test our instrument, plasmid DNA dilutions were amplified with this device; the results demonstrate successful nucleic acid amplification of the …

Different structures of non-viral cationic polymer delivery vehicles, including charge center type, molecular weight and degradability, could significantly affect toxicity, release of nucleic acid and transfection efficiency. Poly(glycoamidoamine)s (PGAAs) contained different carbohydrate and secondary amine moieties and showed high transfection efficiency to different cell lines in a nontoxic manner. The "proton sponge hypothesis" has attempted to relate the buffering capacity to endosomal release of polyethylenimine (PEI) based polyplexes, which could contribute to high transfection efficiency. Secondary amine structures rendered PGAAs buffering capacity around physiological pH. To test the feasibility of the mechanism for PGAAs, new no buffering capacity guanidine or methylguanidine containing poly(glycoamidoguanidine)s (PGAGs) were synthesized. PGAGs formed stable polyplexes with pDNA from N/P (# secondary amine or guanidine group on polymer backbone / # phosphate group on pDNA backbone) ratio 3. PGAG based polyplexes expressed low cytotoxicity and were internalized by 90% of cells at N/P 25. Furthermore, two PGAG based polyplexes showed higher transfection efficiency from N/P 5 to 30 than their PGAA based analogs. These data suggested the low transfection could be due to the difficulties to release pDNA from polyplexes; also, the "proton sponge theory" could not explain the higher transfection efficiency by some PGAGs. Degradation of delivery vehicles could potentially release pDNA in cells and increase transfection efficiency. PGAAs degraded rapidly at physiological conditions and the proposed mechanism was amide hydrolysis. Typically, amide groups are stable and hydrolyze slowly in absence of enzyme. Different models mimicking PGAAs were synthesized to study the fast hydrolysis. Amide groups showed asymmetric hydrolysis. Different hydrolysis behaviors suggested neighboring group participation of two terminal groups to induce rapid amide hydrolysis. These new models could potentially be used to design new polymer delivery vehicles with various degradation profiles. / Master of Science

Nucleic acid therapy holds real promise to offer less severe (lower side effects) as a treatment for life threatening and difficult to treat diseases such as cancer, heart disease or Alzheimer's disease. Theranostic nanomaterials that combine diagnostic imaging and therapeutic delivery, have potential to minimize the amount of time and dosage required for the treatment. This is achieved via delivery of nanoparticles that carry therapeutic payload as well as imaging agents; these agents need to circulate in the body longer due to its (larger) size and selectively accumulate in the tumor regions through the enhanced permeability and retention (EPR) effect. We have designed novel lanthanide (Gd, Tb or La) containing polymers with oligoethyleneamine and lanthanide chelating units to incorporate DNA binding and imaging agent functionality. Protonable amines along the polymer backbone electrostatically interact with DNA and compact it into a nanoparticle. These nanoparticles can be imaged both in vivo (Gd analogues, magnetic resonance imaging) and intracellularly (Tb chelation, fluorescence spectroscopy). Polymers were synthesized via step-growth polymerization to achieve a degree of polymerization of 18-24 for different analogues with varying amine number (three to six, N3-N6) along the backbone. Dynamic light scattering performed on the polyplexes (polymer-DNA complexes) indicate that they are in nanometer size range (50-80nm). All the polymers used to form polyplexes exhibited low toxicity to cultured human Glioblastoma cells (U-87) and showed variable transfection efficiency dependent on structure, comparable to G4 (sold as Glycofect"), a commercial transfection agent previously developed in our lab. This dissertation describes the first studies by the Reineke lab to monitor polyplex formation and destabilization using lanthanide resonance energy transfer (LRET). Polyplexes were formulated with Tb chelated N5 polymer and tetramethyl rhodamine (TMR) labeled pDNA, which are "LRET pairs". We observed decrease in luminescence intensity of Tb polymer (donor) in close proximity of TMR DNA (acceptor) in an intact polyplex at different N/P ratios. However, upon destabilization of polyplexes by addition of salt or heparin solution, the increase in distance between donor and acceptor resulted in increase in the luminescence intensity of Tb polymer. With the LRET technique, we are able to monitor formation and destabilization of polyplexes by monitoring change in luminescence of the donor chromophore (Tb). Polyplexes formulated with non-paramagnetic analogues (La chelated) of N4, polyethyleneimine (PEI) and G4 were studied using NMR to quantify free vs. bound polymer in a formulation. The amount of free polymer was measured by integrating the broad resonances from nanometer-sized particles (polyplexes) with narrow peaks from free polymer chains. This was supported by using an internal reference method to quantify free polymer amount from known internal reference concentration. We observed an increase in the amount of free polymer with N/P ratio for all three systems and both the methods showed comparable results. / Ph. D.

Yes / Magnetic nanoparticles and surface-enhanced Raman scattering (SERS) active nanoparticles were coated with short chain DNA tags. These were then used to identify a target bacterial DNA sequence. The tags function as primers in a standard PCR with the reverse primers and forward primers on the SERS nanoparticles and magnetic nanoparticles, respectively. During the PCR cycles, a composite nanostructure is formed that is both magnetically responsive and SERS active. After magnetic trapping, the intensity of the SERS signal can be related back to the concentration of the target DNA. A test assay was performed that showed a detection limit (based on the signal to noise ratio) of less than 3 zeptomole (41 pg/L). For comparison, a PCR assay based on the standard SYBR Green method was performed. This used the same primers and target DNA and had a detection limit of 10 attomoles (138 ng/L), 3,000 times less sensitive. The work documents the proof of principle study and shows for the first time the use of SERS-NP labels in the quantification of nucleic acid amplification tests and PCR.

Neurodegenerative disease (ND) is a collection of progressive disorders which is marked by gradual degeneration of the central nervous system (CNS). The damage of brain and nervous system causes diminishing of brain and nerve cell which lead to body dysfunction, organ failure, paralysis and ultimately death of a patient. Neurodegenerative diseases affect millions of people worldwide. United States of America is among highest ND prevailing disease countries. Yet the number of cases is underreported because of the complexity of disease diagnosis. With the increase in aging population the rate of ND cases is also increasing. Another important concern is that viral pandemic like COVID-19 may additionally contribute to rise in ND affected population. Currently there is no cure available for ND except a few treatments which decrease some symptoms and decrease disease progression. Early-stage diagnosis of ND is therefore an important and immediate area of research which can improve the quality of life of affected people and help in health management. Many ND has been associated with abnormal expansion of tandem repeat sequence of nucleic acid and other associated protein biomarkers. The discovery of these biomarkers holds promise for improving diagnosis, treatment, and efficacy monitoring. Current detection methods such as neuroimaging techniques, Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) are complex, expensive, require laboratory setup and/or trained operator. Electrochemical biosensing offers a promising portable, inexpensive, and sensitive platform for early and robust diagnosis and healthcare management. In this dissertation a label free electrochemical method is developed to investigate the effect of complex targets for potential genosensing applications and effect of complex biomatrix for immunosensing applications. We first prepare sensing surfaces with three different backbone microprobe nucleic acids to detect length- and sequence-dependent complex secondary structures containing RNA linked to Huntington’s disease, based on the charge transfer resistance of the interface. Then we reported an immunosensing surface using commercially available screen-printed electrode to detect ALS associated biomarker neurofilament light chain in serum samples. Finally, we establish an in-house and cost effective electrochemical immunosensing platform for the detection of ALS biomarker poly-glycine-proline dipeptide repeat in cerebrospinal fluid.

Metal-nucleic acid interaction studies have been gaining attention due to their biological and chemical importance. Nucleic acids are negatively charged bio-polymers and neutralization of their negative charge is essential for the stability and function. In the cells, organic positive ions (positively charged amino acids and polyamines) and some of the metal ions (e.g. Na+, K+, Mg2+...etc) neutralize the charge of nucleic acids. Whereas, interactions of some metal ions (e.g. Cd2+, Hg2+…etc) with nucleic acids destabilize the structure. The stability and conformation of nucleic acids alter due to metal interactions. Further, metal interactions with nucleic acids can bring changes in conformation of ribose, H-bonding and π-π stacking interactions. To understand the metal interactions with nucleic acids, various spectroscopic techniques are being used. However, X-ray crystallographic technique is advantageous over all other spectroscopic techniques since it gives thorough detail of coordination mode and structure. However, crystallization of large molecules like nucleic acids with metals is associated with great difficulty. In order to simplify the problem, nucleic acid constituents and derivatives have been used as model systems for metal-nucleic acid interactions. Nucleic acid constituents and derivatives are multidentate ligands. Moreover, binding mode of metal with nucleic acid constituents and derivatives depends on various factors include pH, temperature, type of metal…etc. Further, understanding of metal nucleic acid interactions can aid to develop new anticancer drugs targeting nucleic acids. For example, cisplatin is a platinum based anticancer drug, which coordinates to N(7) of guanine in DNA brings cell death. There have been several reports in literature on the complexes of metal nucleic acid constituents. However, much more research is warranted for thorough understanding of metal-nucleic acid interactions. On the other hand, nucleic acid constituents and derivatives are used extensively in anticancer drug development. Some of nucleic acid constituent derivatives, 5-Fluro uracil and 6-Mercaptopurine, are currently in use for the treatment of colorectal cancer and leukemia, respectively. Moreover, cisplatin is a platinum based anticancer drug used in the treatment of various types of cancers. However, use of these drugs for long time poses severe side effects and drug resistance. Most of the side effects are due to non bio-compatibility of drugs. To overcome problems associated with present anticancer drugs, bio-compatible metal based anticancer drug development could be an attractive and alternative strategy. To address this, in this study, we report synthesis of a number of new metal complexes of nucleic acid constituents and their derivatives and characterization by various spectroscopic techniques. Also, the interactions of Ni, Cu and Zn ions with various nucleic acid constituents and their derivatives have been elucidated by single crystal X-ray crystallography. Interestingly, Ni, Cu and Zn ions showed various coordination modes to nucleic acid constituents and their derivatives. Further, anticancer studies were carried out for all these complexes in various cancer cell lines. Several complexes showed better cytotoxicity than the well-known drug cisplatin. My thesis work is divided into five parts based on the nature of molecules. I. Synthesis, X-ray crystallographic and anticancer studies on metal (Zn/Ni) complexes of guanine (G) based nucleic acid constituents In order to understand (Zn/Ni) interactions with guanine based nucleic acid constituents and their anticancer activity, several (Zn/Ni) complexes of 5′-GMP, 5′-IMP and hypoxanthine complexes were prepared. The synthesized complexes are (1) [Zn (5′-GMP)]n.11H2O, (2) [Ni (5′-GMP)2 Na2 (μ-OH2)3 (H2O)8].2H2O, (3) [Ni (5′-IMP)2Na2 (H2O)12]n.5H2O and (4) [Ni (hx)2 (H2O)4] Cl2 [Here 5′-GMP = 5′-Guanosine Mono Phosphate, 5′-IMP = 5′-Inosine Mono Phosphate and hx = Hypoxanthine). These complexes were characterized by various spectroscopic and X-ray crystallography techniques. Complex 1: The X-ray structure revealed that zinc is coordinated to 5′-GMP through N(7) position of purine and phosphate moieties, the uncoordinated water molecules are making interesting complicated network of hydrogen bonds in the unit cell. The geometry of zinc coordination centre is distorted tetrahedral. Fascinatingly, zinc exhibited two different coordination environments. In one case, all phosphate oxygens participated in coordination with zinc. In second case, N(7) position of purine and phosphate oxygens participated in coordination with zinc. Moreover, zinc formed a coordination polymer with 5′-GMP. The conformation of ribose changed upon zinc interaction with 5′-GMP from C(3′)-endo to C(2′)-endo, these results suggest that zinc interaction with nucleic acids may change their conformation. Complex 1 is stabilized in solid state by H-bonding and π-π stacking interactions. Complex 2: In complex 2, 5′-GMP is coordinated to nickel through N(7) position of purine but phosphate moiety did not take place in coordination. Two molecules of 5′-GMP and four water molecules coordinated to nickel and formed distorted octahedral geometry. The charge of complex 2 is balanced by sodium coordination to sugar hydroxyl groups and nickel coordinated water molecules. The geometry of sodium coordination centre is distorted octahedral. The conformation of 5′-GMP is altered due to nickel interaction. Moreover, complex 2 is stabilized in solid state by H-bonding and π-π stacking interactions. Complex 3: Nucleotide 5′-IMP also showed similar coordination modes like 5′-GMP towards nickel, where N(7) position of purine participated in coordination with nickel and phosphate moieties did not coordinate to nickel. Two molecules of 5′-IMP and four water molecules participated in coordination with nickel and formed distorted octahedral geometry. Interestingly, the charge of complex 3 is balanced by sodium coordination to sugar hydroxyl moieties. The geometry of sodium coordination centre is distorted octahedral. Moreover, nickel is forming coordination polymer with 5′-IMP. Further, nickel interactions with 5′-IMP brought changes in the conformation of ribose moiety. These results suggest that nickel interactions with nucleic acids may bring changes in their conformation. Interestingly, right hand helical structure formation is observed for complex 3 in crystal structure. Further, the chirality of complex 3 was confirmed by circular dichroism studies. Complex 3 is stabilized by both H-bonding and π-π stacking interactions in solid state. Complex 4: Surprisingly, nickel is coordinated to hypoxanthine through N(9) position of purine in acidic conditions and not through N(7) or N(3). The coordination mode of nickel with hypoxanthine is different from complexes 2 and 3. Two hypoxanthine moieties are coordinated to nickel in axial manner. The geometry of nickel coordination centre is distorted octahedral. Further, complex 4 is stabilized by H-bonding and π-π stacking interactions in solid state. Cytotoxicity studies of complexes 1-4 on various cancer cell lines revealed that complex 1 is better cytotoxic than complexes 2-4. Moreover, complex 1 exhibited comparable cytotoxicity with cisplatin on various cells lines and induced apoptotic cell death. II. Synthesis, structure elucidation and anticancer activity of copper-adeninyl complexes In order to understand copper-adenine interactions and anticancer activity, several copper complexes of adenine derivatives were prepared. Here, most of adenine derivatives used in complex preparation is known as cycline dependent kinase inhibitors. Prepared copper complexes are 1) [Cu (N6-benzyl adenineH)2Cl3 ].Cl.2H2O, 2) [Cu (2-amino-N6-benzyladenineH)2Cl3].(2-amino-N6-benzyl adenineH)2.3Cl.5H2O, 3) [Cu (α-(Purin-6-ylamino)-p-toluenesulfonamide H)2Cl4], 4) [Cu (kinetinH)2 Cl3].Cl.2H2O, 5) [Cu (N-1H-purine-6-yl-alanineH) (H2O) Cl3].H2O, 6) [(Cu (N-1H-purine-6-yl-alanineH)2Cl3).(Cu(N-1H-purine-6-yl-alanineH)Cl)2(μ-Cl)2].Cl.4H2O. All these complexes were characterized by X-ray crystallography and various spectroscopic techniques. Complex 1: Synthesis and X-ray structures of complex 1 were reported in literature. However, anticancer activity of complex 1 is not known. Therefore, it was prepared based on the reported lines to assess the anticancer activity. The anticancer activity of complex 1 was studied on various cell lines. Interestingly, complex 1 exhibited better cytotoxicity than cisplatin in MCF-7 and MDA-MB-231 cell lines. Complex 2: Ligand 2-amino-N6-benzyl adenine is coordinated to copper through N(9) of purine. In addition, two uncoordinated 2-amino-N6-benzyl adenine, three chloride and five water molecules are making it as a co-complex with uncoordinated ligands. The copper coordination centre adopted distorted trigonal bipyramidal geometry [3+2] with τ = 0.671 (α-β/60, where α, β are two greatest valence angles of coordination centre). Further, complex 2 is stabilized in solid state by both H-bonding and π-π stacking interactions. H-bonding is observed between N-H···Cl. Uncoordinated water molecules formed six-member rings with H-bonding network. The π-π stacking interactions are observed between phenyl and purine moieties. Complex 2 exhibited better cytotoxicity than 2-amino-N6-benzyl adenine and copper salt. Complex 3: Ligand α-(2-Amino purin-6-ylamino)-p-toluene sulfonamide is coordinated to copper through N(9) position and protonation is observed at N(3) position. Two molecules of α-(2-Amino purin-6-ylamino)-p-toluene sulfonamide and four chloride ions are forming a distorted octahedral geometry with copper. Complex 3 is stabilized by N-H···Cl and N-H···O H-bonding. Further, complex 3 exhibited better cytotoxicity than cisplatin in U251 cells. Complex 4: Kinetin is coordinated to copper through N(9) position of purine. Protonation is observed on N(3) position and balanced the charge of complex 4. Two molecules of kinetin and three chloride moieties are coordinated to copper and forming distorted trigonal bipyramidal geometry [3+2] with τ = 0.431. Moreover, complex 4 is stabilized by both H-bonding interactions and π-π stacking interactions. The H-bonding of complex 4 is observed between N-H···Cl and C-H···Cl. The π-π stacking interactions are observed between furanyl aromatic ring and imidazole ring of purine. Complex 4 exhibited better cytotoxicity than kinetin and copper salt. Complex 5: The N-1H-purine-6-yl-alanine is coordinated to copper through N(9) position of purine. Complex 5 crystallizes in the monoclinic space group P21 with Z=4. One molecule of N-1H-purine-6-yl-alanine, two chloride ions and one water molecule coordinated to copper. The geometry of copper coordination centre is distorted trigonal bipyramidal [3+2] with Cu(1) τ1 = 0.613 and Cu(2) τ2= 0.671. Protonation is observed on N(3) position. Complex 5 is stabilized by both H-bonding and π-π stacking interactions. The H-bonding of complex 5 is observed between N-H···Cl and C-H···Cl. The π-π stacking interactions are observed between imidazole moieties. Moreover, complex 5 exhibited better cytotoxicity than N-1H-purine-6-yl-alanine and copper salt. Complex 6: Complex 6 is a co-complex, where two different complexes are co-crystallized. The crystal structure of complex 6 indicate that geometry of Cu(1) and Cu(2) coordination centre are distorted trigonal bipyramidal [3+2] with τ1 = 0.3261 and τ2 = 0.8, respectively. Two molecules of N-1H-purine-6-yl-alanineH are coordinated to Cu(2) through N(9) position of purine. The N-1H-purine-6-yl-alanineH ligands are arranged in geometry in trans manner with respect to axis passing through the N(9) atom and copper. Whereas, in second co-complex two N-1H-purine-6-yl-alanineH are coordinated to Cu(1) through N(9) and N(3) position of purine. Both Cl(1) and Cl(3) coordinated to copper are forming a bridge between copper. In addition, one uncoordinated chloride and two water molecules are present in the unit cell. Complex 6 is stabilized in crystalline state by both H-bonding and π-π stacking interactions. Complex 6 exhibited better cytotoxicity than complex 5, N-1H-purine-6-yl-alanine and copper salt on various cell lines. III. Synthesis, structure and anticancer activity of zinc complexes of adenine derivatives In order to understand zinc interaction with adenine and their anticancer activity, several zinc complexes of adenine derivatives were prepared. The prepared complexes are (1) [Zn (N6-benzyladenineH).Cl3].2H2O, (2) [Zn2 (μ -N6-benzyladenine)2( μ-H2O)2(H2O)4].(OTf)4.H2O, (3) (N6-benzyl adenineH2) [ZnCl4].2H2O, (4) [Zn (2-Amino-N6-Benzylpurine)Cl3).2-Amino-N6-BenzylpurineH).EtOH, (5) (2-Amino-N6-(3-picoyl)purineH2)[ZnCl4].H2O, (6)(2-Amino-N6-(3-picoyl)purineH2)[ZnCl4].HCl, (7) (2-Chloro-N6-(3-picoyl) purineH2) [ZnCl4].H2O, (8) ((α-Purine-6-ylamino)-p-toluene sulfonamide H)2[ZnCl4].2HCl.2H2O. Complex 1: The N6-benzyl adenine is coordinated to zinc through nitrogen atom N(7) of purine. One molecule of N6-benzyl adenine and three chloride ions are coordinated to zinc and forming distorted tetrahedral geometry. Interestingly, the nitrogen atom N(1) of purine is protonated. Complex 1 exhibited strong H-bonding interactions between N-H···O, N-H···Cl and N-H···N. The complex 1 showed better cytotoxicity than N6-benzyl adenine and ZnCl2. Complex 2: The N6-benzyl adenine formed a dimeric complex with zinc at neutral pH. Complex 2 crystallizes in the triclinic space group P-1with Z=1. Two Zn metal centres are bridged by two molecules of N6-benzyl adenine through nitrogen atoms N(3) and N(9) of purine forming a di-nuclear complex, further two zinc centres is bridged by two water molecules and other two water molecules on the other side completing the octahedral coordination for the Zn. Complex 2 is stabilized in crystalline state by H-bonding interactions. The H-bonding of complex 2 is observed between O-H···O and N-H···O. Complex 2 exhibited better cytotoxicity than N6-benzyl adenine and ZnCl2 on various cell lines. Complex 3: The N6-benzyladenine is not coordinated to the Zn metal at acidic pH and forms an ion-pair complex. Ion-pair complex 3 crystallizes in the monoclinic space group Cc with Z=4. The protonation is observed at N(1) and N(9) atoms of N6-benzyl adenine. The positive charges on N6-benzyl adenine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Alternative arrangement of cation and anion arrangement is observed in complex 3. Water channel formation is observed between cation and anion arrangement. Moreover, complex 3 is stabilized by H-bonding and π-π stacking interactions. H-bonding is observed in complex 3 between N-H···Cl, O-H···Cl and N-H···O. The π-π stacking interactions in complex 3 are observed between benzyl six-membered aromatic ring and purine six-membered rings. Complex 3 exhibited better cytotoxicity than N6-benzyl adenine and ZnCl2 in various cell lines. Complex 4: Ligand 2-amino-N6-benzyl adenine resulted in a different structure from N6-benzyl adenine with zinc. One molecule of 2-amino-N6-benzyl purine is coordinated to zinc through nitrogen atom N(7) of purine. Surprisingly, one uncoordinated positively charged 2-amino-N6-benzyl purineH is present in the asymmetric unit, which is balancing the charge of zinc complex 4. Protonation is observed on N(3A) atom. Interestingly, tautomeric proton is located on coordinated purine of N(9) atom and uncoordinated purine of N(7A) atom. Geometry of ‘Zn coordination centre’ is distorted tetrahedral. Complex 4 is stabilized by H-bonding and π-π stacking interactions. The H-bonding interaction in complex 4 is observed between N-H···O and N-H···Cl. The π-π stacking interactions are observed between five-member aromatic rings and six-membered aromatic rings. Complex 4 exhibited better cytotoxicity than 2-amino-N6-benzyl purine and ZnCl2 in various cell lines. Complex 5: 2-Amino-N6-(3-picoyl) purine forms an ion-paired complex with zinc at acidic pH. The protonation in 2-Amino-N6-(3-picoyl) purine is observed at N(3) of the purine and picolyl N(14). The positive charge of 2-Amino-N6-(3-picoyl) purine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Moreover, complex 5 exhibited both H-bonding interactions and π-π stacking interactions. The H-bonding interactions are observed between N-H···Cl, N-H···N, O-H···Cl, N-H···O and C-H···N. One uncoordinated water molecule is present in unit cell, which is involved in H-bonding with both ions. The π-π stacking interactions are observed between purine five-membered rings and purine six-membered ring. Complex 5 exhibited better cytotoxicity than cisplatin in HeLa and MDA-MD-231 cells. Complex 6: 2-Amino-N6-(3-picoyl) purine formed similar structure of complex 5 in strong acidic conditions. Complex 6 exhibited both H-bonding and π-π stacking interactions. The H-bonding in complex 6 is observed between N-H···Cl and N-H···N. In complex 6, the π-π stacking interactions are observed between pyridyl six-membered rings and purine six-membered rings. Purine-Purine stacking interactions are observed between purine six-membered ring and five-membered rings. Complex 6 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7, MDA-MB-231 and HeLa-Dox cells. Interestingly, complex 6 arrested (G2/M phase) cell cycle in HeLa and MCF-7 at higher concentration and induced apoptosis. Complex 7: 2-chloro-N6-(3-picoyl) purine formed ion-pair complex with zinc. The protonation in 2-chloro-N6-(3-picoyl) purine is observed on N(9) of purine and N(14) of picolyl atoms. The positive charge of 2-chloro-N6-(3-picoyl) purine is neutralized by the presence of two chloride ions in [ZnCl4]2-. Complex 7 is stabilized by both H-bonding and π-π stacking interactions. The H-bonding is observed between N-H···Cl, O-H···Cl and N-H···O in complex 7. The π-π stacking interactions are observed between pyridyl six-membered ring and six-membered ring of purine. Complex 7 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7, U251 and HeLa-Dox cells. Complex 8: (α-Purine-6-ylamino)-p-toluene sulphonamide formed ion-pair complex with zinc. Ion-pair complex 8, crystallizes in the triclinic space group P-1 with Z=4. The protonation on (α-Purine-6-ylamino)-p-toluene sulfonamide is observed at N(9) and N(1) atoms of purine. The positive charge of the ligand is neutralized by two chloride ions present in [ZnCl4]2 -. The H-bonding is observed between N-H···Cl, O-H···N, N-H···O and O-H···Cl. The π-π stacking interactions are observed between benzyl rings of benzene sulfonamide moieties. Complex 8 exhibited better cytotoxicity than cisplatin in HeLa, MCF-7 and HeLa-Dox cells. Moreover, these complexes induced apoptotic cell death as revealed by Annexin V/PI assay, FACS and microscopy analysis. IV. Synthesis, structure and cytotoxicity studies of zinc complexes of uracil-1-acetic acid and N6-adeninebutyric acid To understand the zinc interactions with nucleic acid constituent derivatives and their anticancer activity, zinc complexes of uracil-1-acetic acid and N6-adeninebutyric acids were prepared. (1) [Zn (uracil-1-acetato)2 (H2O)4] and complex (2) [Zn (N6-adeninebutyric acid)2 (H2O)2]) were characterized by X-ray crystallography and various spectroscopic techniques. The X-ray structures showed acetate moiety coordination to zinc rather than purine and pyrinidine moities. The geometry of zinc coordination centre is distorted octahedral. Complexes 1 and 2 are stabilized by non-covalent interactions. Anticancer studies of these complexes showed better cytotoxicity than cisplatin in MDA-MB-231cells. V. Copper (II) complexes of 6-mercaptopurine, hypoxanthine and uracil-1-acetic acid: Synthesis, structures, antioxidant and potent anticancer activity To delineate copper interactions with purine and pyrimidine derivatives and anticancer activity, several copper complexes of 6-mercaptopurine, hypoxanthine and uracil-1-acetic acid were prepared. The prepared complexes are (1) [Cu (6-MP) (bpy) Cl2], (2) [Cu (hx) (phen) Cl2].H2O and (3) [Cu (bpy)2 (uracil-1-acetato)].6H2O)] (bpy = 2, 2′-bipyridine, phen = 1, 10-phenanthroline, 6-MP = 6-Mercapto Purine and hx = hypoxanthine). All these complexes were chracterized by various spectroscopic and X-ray diffraction techniques. Complexes 1 and 2 crystallize in the monoclinic space groups Cc and C2/c, respectively with eight molecules in the unit cell. All the complexes 1-3 adopt distorted trigonal bipyramidal geometry. Surprisingly, most potent coordination sites of sulfur in 6-MP and acetato in uracil-1-acetato did not participate in coordination with copper. In complexes 1 and 2, the N(7) position of purine and the N(3) position of pyrimidine in complex 3 are coordinated with copper. All these complexes 1-3 are stabilized by non-covalent interactions in solidstate. Anticancer studies showed better cytotoxicity for copper complexes than cisplatin, 6-meracptopurine and temozolomide in various cell lines. Interestingly, copper complexes of 6-MP and hypoxanthine showed antioxidant activity and reduced ROS level in cells. In contrast, copper complex of uracil-1-acetic acid produced ROS in cells. In contrast, copper hypoxanthine showed better cytotoxicity than cisplatin in HeLa-Dox cells. All these complexes induced apoptotic cell death. In summary, we studied the interaction of metal-nucleic acid constituents and derivatives by X-ray crystallography. We found new coordination modes for Ni, Cu and Zn towards various nucleic acid constituents and derivatives. Some of these complexes showed better cytotoxicity than well known anticncer drugs cisplatin, 6-meracptopurine and temozolomide. Complex [Cu (hx) (phen) Cl2].H2O showed better cytotoxicity than cisplatin in doxorubicin resistant (HeLa-Dox) cells. These complexes induced apoptotic cell death in various cancer cells. All in all, the results of present studies/findings could form a potential lead for the development of newer anticancer therapeutics.

<p dir="ltr">Since the beginning of the HIV/AIDS epidemic, 85.6 million people worldwide have become infected with HIV; more than half of whom have died from AIDS-related complications.[1] Sustained viral suppression below the clinically relevant threshold (1000 copies per mL) with highly active antiretroviral therapy (HAART) has proven effective at managing and prolonging the life expectancy of people living with HIV (PLHIV). However, in 2022, 11.3 million PLHIV had still not achieved viral suppression and may become susceptible to both HIV transmission and a variety of opportunistic infections. Of particular importance is the complex issue of patient non-compliance in global HIV management due to social, economic, behavioral, and healthcare access barriers, potentially disconnecting many PLHIV from the HIV care continuum. Therefore, to boost patient engagement in clinical care and to improve overall patient outcomes, new approaches to viral load monitoring practices need to be developed to increase access, particularly in regions of high HIV prevalence.</p><p dir="ltr">Nucleic acid amplification tests (NAATs) have emerged as potent tools for monitoring viral load, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) being recognized as the benchmark due to its sensitivity and ability for real-time quantification enabled by fluorescence signal emission. Nevertheless, RT-qPCR is burdened by drawbacks including extended processing times, high operational costs, and the requirement for specialized laboratory facilities. In this study, we propose a novel method for HIV-1 viral load monitoring by integrating reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) with real-time particle diffusometry (PD). This approach allows for the continuous monitoring of changes in the diffusion of 400 nm fluorescent particles during RT-LAMP amplification, targeting the <i>p24</i> gene region of HIV-1 RNA. This enables the real-time detection of amplification curves, achieving a detection sensitivity in water samples as low as 25 virus particles per μL within a short duration of 30 minutes. Additionally, to address challenges related to amplification inhibition in complex human specimens, we developed a power-free sample processing system specifically designed for extracting HIV-1 RNA from both whole blood and plasma.Top of FormBottom of FormThis system modifies a commercially available spin-column protocol by integrating a syringe device and handheld bulb dryer, thus eliminating the requirement for a centrifuge. The adaptation allows for the completion of the entire extraction procedure, encompassing viral lysis, RNA capture, washing, and elution of purified HIV-1 RNA, within a timeframe of less than 16 minutes. Subsequent analyses, including RT-LAMP and RT-qPCR, demonstrate a limit of detection of 100 copies per μL and an average RNA recovery of 32% (for blood) and 70% (for plasma) in the elution fraction. Further investigations emphasize the significant presence of purified RNA in the spin column volume (termed as dead volume), and the cumulative recovered RNA copies align with those obtained using the gold standard centrifugation extraction method. Ultimately, we incorporated the real-time quantitative PD-RT-LAMP assay onto a field-compatible handheld portable platform suitable for field use, featuring built-in quality control measures. This platform enables sample-to-answer viral load testing near the point of care (POC). Subsequently, we undertook essential preparatory steps, such as reagent drying to obviate the need for cold storage, initial device calibration, and hands-on training of laboratory personnel regarding device operation, to validate device performance within a cohort of individuals living with HIV (PLHIV). These innovations facilitate quick and comprehensive viral load determination, offering promise for enhanced HIV management and patient care</p>

A peptide nucleic acid (PNA) encoded 1296 member peptide library was synthesised and incubated with a variety of cell types. Library members entering cells were extracted, hybridised onto DNA microarrays and the peptide identity was determined via deconvolution. Global consensus analysis highlighted the tetrapepide, Glu-Llp- Glu-Glu (Llp is 6-hexamine-N-aminoacetic acid), a surprise in view of the basic residues typically observed in cell penetrating peptides. When evaluated, Glu-Llp- Glu-Glu revealed cellular uptake comparable to a known basic peptide (tetraLlp). In depth delineation via clustering analysis allowed assessment of differential cellular uptake, with the identified peptides showing clear cellular specificity. This was verified by peptide synthesis and cellular uptake analysis by flow-cytometry, and in all cases an endocytic uptake mechanism was confirmed. This approach establishes a strategy for the identification of short peptides as tools for selective delivery into specific cell types. The incubation of a 10,000 member PNA-encoded peptide library with D54 and HEK293T transfected with CCR6 cells followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new ligands for integrins and G-protein coupled receptors and offers a novel approach to ligand discovery allowing the comparative analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between various cell types. In addition, this work included the development of a novel method for the indirect amplification of a PNA library by amplification of a complementary DNA library hybridised to the PNA. The generation of 10,000 defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification of immobilised DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3875; 10,000 defined oligonucleotides) were used to validate the process, with sequences verified by selective hybridisation to a complementary DNA microarray with DNA sequencing demonstrating error rates of ca ≈ 0.2%. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA primers, while the DNA-arrays can be used as “factories” allowing specific DNA oligonucleotide pools to be generated with or without masking. This study also demonstrated a significant variance observed between the sequence frequencies found via Solexa sequencing compared to microarray analysis.

Current chemotherapeutics exhibit debilitating side effects as a result of their toxicity to healthy tissues. Reducing these side effects by developing chemotherapeutics with selectivity for cancer cells is an active area of research. Phototherapy is one promising modality for selective treatment, where drug molecules are “turned on” when irradiated with light, reducing damage to healthy tissues by spatially restricting the areas exposed to irradiation. A second approach to improve selectivity is to exploit the differences in cancerous versus healthy cells, such as increased metabolism and/or upregulation of cell surface receptors. Ruthenium(II) polypyridyl complexes are candidates for phototherapy due to their highly tunable photophysical and photochemical properties. The addition of strain to the metal center is a general approach used to render complexes susceptible to light-induced ligand loss. Upon ejection of a ligand, the Ru(II) center is capable of covalently binding biomolecules within cells to produce a cytotoxic effect. The ligands surrounding the metal center are amenable to chemical modification through the incorporation of pendent functional groups as chemical “handles”, allowing for different directing molecules to be attached. Nucleic acids are important targets for drug discovery, and the development of selective probes to either visualize or selectively damage nucleic acids within the cell is an ongoing area of research. Specifically, G-rich regions are abundant in the human genome, and the presence of G-quadruplexes in telomeres and promoter regions of oncogenes make them potential therapeutic targets. Ru(II) complexes are known to bind nucleic acids, and some have been shown to induce and/or stabilize G-quadruplex Structures. Multiple series of Ru(II) compounds have been synthesized and tested to improve the functional range for Ru(II) complexes for in vivo applications, where they act as “light switches” for DNA. These molecules are “off” when in an aqueous environment but turned “on” in the presence of DNA. Several hit compounds were identified that showed selectivity for specific G-quadruplex structures.

Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.