Novel secondary structures of DNA; and development of a sensitive methodology for capturing DNA/RNA G-quadruplexes from living Drosophila salivary glands

Work reported in this thesis, from three independent projects, highlights: first, a novel DNA secondary structure fold from a neurodegenerative disease-linked repeat sequence; second, a new approach for assembling and reversing a long and 1- dimensional DNA nanostructure. The third and most substantial project reports the development of and biological results from a highly selective and sensitive approach for in vitro and in vivo tagging of DNA and RNA G-quadruplexes. In the first project, a wholly novel higher-order fold of DNA, named as “iCD-DNA”, was discovered and characterized. iCD-DNA was found to be formed uniquely by a hexanucleotide repeat expansion sequence, d(C2G4)n, located at the 5’ UTR of the C9orf72 gene, causally linked to multiple neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). It was found that incubating d(C2G4)n under mildly acidic conditions and in the presence of non-quadruplex supporting cations (e.g. Li+, Mg2+) gave rise to a distinctive higher order structure whose most striking feature was an inverted circular dichroism (CD) spectrum, distinguishable from the inverted CD spectra of either a left-handed duplex (“Z-DNA”) or a left-handed G-Quadruplex (“Z-GQ”). On the basis of CD spectroscopy, gel mobility and chemical footprinting, structural models were proposed for iCD-DNA. In the second project, a new strategy for creating a long (~200-300 nm) and reversible 1-Dimensional DNA nanostructure/ nanowire (1DDN), named “(TQs)n”, was designed and carried out. “(TQs)n” incorporates a hybrid of DNA triple and quadruplex helices. In this design, a novel approach for joining together DNA helices (called guanine-rich “glue junctions”) was proposed and demonstrated. In the third project, a highly specific and sensitive methodology for uniquely biotin-tagging DNA/RNA G-quadruplexes (by way of their intrinsic peroxidase activity while complexed with heme) was deeply characterised, first, in vitro, and then applied to tag and pull down G-quadruplex forming RNAs and DNAs from living Drosophila larval salivary glands. Preliminary-sequencing data, so obtained, provided initial insights for the potential occurrence of G-quadruplexes in living cells but needs detailed future investigation

A Commemorative Issue in Honor of Professor Nick Hadjiliadis: Metal Complex Interactions with Nucleic Acids and/or DNA

This Special Issue of the International Journal of Molecular Science comprises a comprehensive study on “Metal Complex Interactions with Nucleic Acids and/or DNA”. This Special Issue has been inspired by the important contribution of Prof. Nick Hadjiliadis to the field of palladium or/and platinum/nucleic acid interactions. It covers a selection of recent research and review articles in the field of metal complex interactions with nucleic acids and/or DNA. Moreover, this Special Issue on "Metal Complexes Interactions with Nucleic Acids and/or DNA" provides an overview of this increasingly diverse field, presenting recent developments and the latest research with particular emphasis on metal-based drugs and metal ion toxicity

Canonical and non-canonical DNA methylome dynamics during vertebrate development

Upon fertilisation of vertebrate embryos, the epigenomes of the responsible gametes need to be reconfigured into a state that is compatible with totipotency and zygotic transcriptional programs. Furthermore, the epigenomes of differentiating cells then need to be remodelled again in order to form the complex structures of the body, such as the vastly intricate nervous system. This includes, but is not limited to, the remodelling of DNA methylation, the most abundant DNA modification in vertebrates with critical roles in embryogenesis and neurodevelopment. In mammals, methylation of cytosines in cytosine-guanine dinucleotides (mCG) is almost completely erased after fertilization before it is re-established during gastrulation. Similarly, methylation of cytosines outside the CG context (mCH; H = A,T,C) is diluted in the early mammalian embryo before it is re-established mainly in the nervous system. However, in non-mammalian vertebrates, it appears that no global erasure of mCG takes place, raising questions about their propensity for transgenerational epigenetic inheritance. Additionally, the conservation of mCH in non-mammalian vertebrates is largely unexplored. In this thesis, I look to expand our knowledge on the developmental dynamics, evolutionary conservation and the molecular components of DNA methylome remodelling in vertebrates by studying methylome dynamics in two distantly related teleost species (ray-finned, protruding jawed fish). I functionally explore how DNA methylation is regulated during the development of zebrafish (Danio rerio), medaka (Oryzias latipes), and zebrafish-medaka hybrids, in both the CG and CH context. I employ CRISPR/cas9 technology, whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and RNA sequencing (RNA-seq), to interrogate a wide range of developmental time points and adult tissues. Overall, I have: i) developed a system to functionally test for regulators of developmental DNA methylation; ii) revealed a novel form of developmentally remodelled mCH in zebrafish and medaka which is deposited by the teleost specific DNMT3BA enzyme, iii) demonstrated evolutionary conservation of mammalian-like mCH features in the developing zebrafish nervous system, and iv) shown that DNA methylome dynamics in medaka and zebrafish embryos are highly comparable and compatible during the first 24 hours of zebrafish-medaka hybrid development. Altogether, this work greatly expands our understanding of the form and function of a critical DNA modification during development

Transcriptome analysis of bovine day 16 conceptus derived after transfer of blastocyst from somatic cell nuclear transfer or in vitro production

In vitro embryo production (IVP) and somatic cell nuclear transfer (SCNT) have been used as tools of assisted reproductive technology to produce bovine pre-implantation embryos independent of the maternal environment. However, the embryonic and fetal losses after transfer of SCNT and IVP derived embryos is higher compared to the in vivo (AI) counterparts. This may be associated with the alterations in the molecular signatures and pathways at any stage of embryonic and /or fetal development. Therefore, to identify the molecular changes that could occur at day 16 SCNT and IVP derived embryos, large scale transcriptomic analysis was performed using Affymetrix-Bovine Genome Array. For this, day 7 blastocysts derived from SCNT, IVP and AI were transferred to oestrus synchronized Simmental heifers. Recipients were then slaughtered at day 16 of gestation and conceptuses were retrieved. Following morphological examination, filamentous embryos with visible embryonic disc were subjected to global tanscriptome analysis. The result demonstrated comparable in vivo development rate in SCNT (72.7%), IVP (62.2%) and AI (77.3%) embryo groups. However, considerable reduction in the trophoblast elongation size was observed in SCNT (93.3mm) compared to IVP (186.6mm) and AI (196.3mm) derived embryos. In addition, more than 20% of SCNT (10.7 mm ± 1.08) and IVP (20.1 mm ± 0.15) conceptuses had tubular shape, suggesting a delay in recapitulating filamentous morphology. Gene expression profiling analysis revealed that the transcript levels of 477 genes, which are involved in various pathways including arginine and proline, glycerolipid and fatty acid metabolism, were significantly altered in SCNT embryos compared to AI. Similarly, 365 genes were differentially expressed in IVP embryos compared to AI. Thus, several canonical pathways including TNRF-1 and tight junction signalling pathways were affected in IVP derived conceptuses. To predict whether the altered transcripts were associated with pre-elongation in vitro culture environment or errors in transcriptional reprogramming, unique or commonly differentially expressed genes were analyzed in SCNT and IVP embryos compared to AI or donor cells (fibroblast). Accordingly, 71 transcripts including (FOLR1, MYO1B, RCN2, H2AFJ, HSPB1 and GATM) were found to be not transcriptionally reprogrammed as their expression resembled more the donor cells than AI embryos. The remaining transcripts were either partially or incompletely reprogrammed. In addition, quantitative real time PCR (qPCR) based expression profiling of candidate transcripts in developmentally delayed SCNT or IVP embryos showed low mRNA levels of IFNt, FGFR2, CLDN1 and ARHGEF2 in developmentally lagging IVP and SCNT embryos compared to their respective elongated counterparts. In conclusion, the present study identified deviation in elongation size, gene expression and the corresponding molecular pathways in day 16 SCNT and IVP conceptuses compared to their AI counterparts which may subsequently be associated with fetal development.Transkriptom-analyse von bovinen 16 Tage alten Embryonen, gewonnen durch den Transfer von Blastozysten aus klonierten somatischen Zellen sowie der in vitro Produktion In vitro Embryo Produktion (IVP) und somatischer Kerntransfer (SCNT) sind Werkzeuge der assistierten Reproduktionstechnologien und finden ihren Einsatz um bovine Präimplantations- embryonen unabhängig von der mütterlichen Umwelt zu erzeugen. Allerdings sind embryonale und fetale Verluste nach dem Transfer von SCNT und IVP gewonnenen Embryonen höher im Vergleich zu in vivo (AI) erzeugten Embryonen. Dies kann mit den Veränderungen der molekularen Signaturen sowie Signalwegen in den unterschiedlichen Stadien der embryonalen und/oder fetalen Entwicklung zusammenhängen. Um molekulare Veränderungen zu identifizieren, die am Tag 16 von SCNT und IVP gewonnene Embryonen auftreten können, wurde mit Affymetrix-Bovine Genome Arrays eine Transkriptomanalyse durchgeführt. Hierzu wurden Tag 7 Blastozysten von SCNT, IVP und AI erzeugten Embryonen in Östrus synchronisierte Fleckviehfärsen übertragen. Am Tag 16 der Trächtigkeit wurden die Rezipienten geschlachtet und die Embryonen entnommen. Nach morphologischen Untersuchungen wurden filamentöse Embryonen mit sichtbarer Keimscheibe einer globale Tanskriptomanalyse unterzogen. Das Ergebnis zeigte in den verschiedenen Embryogruppen SCNT (72,7%), IVP (62,2%) und AI (77,3%) eine vergleichbare in vivo Entwicklung. Allerdings konnte eine erhebliche Verringerung in der Größe der Trophoblasten Elongation in SCNT (93,3 mm) im Vergleich zu IVP (186,6 mm) und AI (196,3 mm) Embryonen beobachtet werden. Darüber hinaus wiesen mehr als 20% der SCNT (10,7 mm ± 1,08) und IVP (20,1 mm ± 0,15) Embryonen eine Röhrenform auf, was auf eine verzögerte rekapitulierte filamentöse Morphologie hindeutet. Die Auswertung der Transkriptomanalyse zeigte beim Vergleich von SCNT mit AI 477 signifikant unterschiedlich expremierte Gene, die in verschiedenen Signalwegen beteiligt sind, einschließlich Arginin und Prolin, Glycerolipid und Fettsäure-Metabolismus. Des Weiteren wurden 365 signifikant unterschiedlich exprimierte Gene beim Vergleich von IVP Embryonen mit AI Embryonen identifiziert. Relevante Signalwege dieser Gene waren unter anderem TNRF-1 und Tight-Junction Signalisierung. Um festzustellen, ob die veränderten Transkripte mit der in in vitro Kultur bedingten Präelongation oder mit Fehlern der transkriptionellen Reprogrammierung assoziiert sind, wurden einzigartige oder häufig unterschiedlich exprimierte Gene in SCNT und IVP Embryonen gegenüber AI oder Donorzellen (Fibroblasten) analysiert. Dementsprechend zeigten 71 Transkripte einschließlich FOLR1, MYO1B, RCN2, H2AFJ, HSPB1 und GATM keine transkriptionelle Reprogrammierung, da deren Expressionprofil mehr dem der Donorzellen als dem der AI Embryonen ähnelte. Die restlichen Transkripte waren entweder teilweise oder vollständig reprogrammiert. Zusätzlich, zeigten auf quantitative Real Time PCR (qPCR) basierende Kandidatengenexpressionsprofile in entwicklungsverzögerten SCNT oder IVP Embryonen niedrigere mRNA Spiegel in IFNtau, FGFR2, CLDN1 und ARHGEF2 im Vergleich zu ihren elongierten Gegenstücken. Schlussfolgernd konnten mit dieser Studie Abweichungen in den Elongationsgrößen, den Expressionsprofilen und den entsprechenden molekularen Signalwegen in Tag 16 SCNT und IVP produzierten Embryonen im Vergleich zu AI produzierten Embryonen beobachtet werden. Diese Ergebnisse könnten in Zusammenhang mit den weiteren fötalen Entwicklung gebracht werden

Raman spectroscopy of biological tissue for application in optical diagnosis of malignancy

The use of Raman spectroscopy in the detection and classification of malignancy within the human larynx and lymph nodes of the head and neck has been evaluated. Currently histopathology is considered the diagnostic gold standard. The potential for Raman spectroscopy to be used as an in vivo diagnostic tool in the detection of dysplasia and malignancy has been demonstrated. A consensus opinion from three expert histopathologists has been obtained and spectral diagnostic models developed by correlation with these results. The ability of Raman spectroscopy to differentiate between disease entities and normal tissue within the larynx has been shown. Raman spectroscopy was able to identify non-neoplastic vocal cord mucosa (sensitivity 85 %, specificity 95%) from laryngeal mucosa showing neoplastic change (sensitivity 95 %, specificity 85%) with an increase in sensitivity to 89% for the non-neoplastic tissue and a reduction to73% in tissues showing neoplastic changes after cross-validation. For the first time benign changes in the structure of vocal cords such as those exhibiting hyperkeratosis and hyperplasia, where also identified with sensitivity of 97.9% for tissue exhibiting hyperplasia/hyperkeratosis and 100% for normal squamous cell epithelium. Research into the ability of Raman spectroscopy to interrogate lymphoid tissue in order to differentiate reactive nodes (sensitivity 90 %, specificity 88%) from those containing cancer (sensitivity 88 %, specificity 90%) was successful and fully independently validated. This work was further developed and the efficacy of Raman spectroscopy in differentiating between squamous cell carcinoma (sensitivity 76%, specificity 95%), adenocarcinoma (sensitivity 93 %, specificity 99%), Hodgkin‘s lymphoma (sensitivity 80%, specificity 90%) and reactive lymph nodes (sensitivity 81%, specificity 88%) was shown. This model was also independently cross-validated by node producing further improvements to give a spectral performance of sensitivity/specificity for SCC of 75/97%, adenocarcinoma 100/99%, Hodgkin‘s lymphoma 83/92% and reactive lymph nodes 85/86%.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

STRUCTURAL AND FUNCTIONAL STUDIES OF AN ATYPICAL OMPR/PHOB TRANSCRIPTIONAL REGULATOR, CHXR, FROM CHLAMYDIA TRACHOMATIS

Chlamydia infections have an immense impact on public health and are associated with diverse disease manifestations including atherosclerosis, blindness, and sterility. The chlamydial developmental cycle is intrinsically linked with the ability of the organism to cause disease. The mechanisms that regulate the developmental cycle are poorly understood; however, transcription appears to play a governing role. An OmpR/PhoB subfamily response regulator termed ChxR exhibits expression patterns that indicate an important role during the developmental cycle. Previously, ChxR was demonstrated to interact with its own promoter and facilitate the transcriptional activation of the chxR gene. To begin to understand the functional role of ChxR, I identified the DNA sequence recognized by ChxR to identify its gene targets. Primarily using gel mobility shift assays, I determined that ChxR interacts with, and has differential affinity for six binding sites in the chxR promoter region. Using the DNA sequences from these binding sites, I elucidated the ChxR cis-acting recognition sequence. Additionally, I was interested in elucidating the ChxR mechanism of transcriptional activation. Usually as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Dimer formation facilitates an interaction of the effector domain interaction with DNA and transcriptional machinery to regulate transcription. ChxR is an atypical OmpR/PhoB response regulator because it is active in the absence of phosphorylation. We hypothesized that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR protein are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. Using biochemical techniques, I demonstrated that ChxR forms homodimers through the receiver domain and the effector domain interacts with DNA similar to phosphorylation-induced and transcriptionally active OmpR/PhoB response regulators. Additionally, the structures of the two domains were solved to direct functional studies to identify the residues important in homodimer formation, interaction with DNA, and interaction with RNA polymerase machinery. Both structures had unique features that are not found in other OmpR/PhoB subfamily members. The combination of these results suggests that ChxR is a member of the OmpR/PhoB subfamily, although many of the characteristics of the subfamily are not shared in ChxR

Structural Mechanism of Substrate Specificity In Human Cytidine Deaminase Family APOBEC3s

APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy

Vibrational Imaging at the Nanoscale: Surpassing the Diffraction Limit Using Tip-Enhanced Raman Spectroscopy

A deep understanding of the chemical composition of surfaces, interfaces or nanoscale structure with a high spatial resolution is an important goal in nanoscience and nanotechnology. Structural information can be collected using a variety of high spatial resolution techniques such as atomic force microscopy (AFM), scanning tunneling microscopy (STM), scanning electron microscopy (SEM), or transmission electron microscopy (TEM). Nevertheless, these methods do not offer molecular information such as vibrational spectroscopy techniques that allow one to collect molecular or lattice vibrations yielding to a precise picture of the molecular interactions in bulk materials as well as in surfaces and interfaces. Unfortunately optical spectroscopy techniques are limited in terms of spatial resolution and sensitivity due to the poor signal/noise ratio of the localized measurement. Surface- and tip-enhanced Raman spectroscopy (SERS and TERS) are advanced spectroscopic techniques, which are becoming widely used and show a great potential for the structural characterisation of biological systems. Surface-enhanced spectroscopy (SERS) was developed to improve the sensitivity of the chemical measurements by using rough silver or gold surfaces. The challenge of the simultaneous improvement of the spatial resolution and sensitivity was addressed by combining high resolution optical microscopy with the high sensitivity of surface-enhanced spectroscopy and was termed tip-enhanced Raman spectroscopy (TERS). In this thesis, gap-mode TERS is developed for the study of a variety of materials. TERS is used in conjunction with gold nanoplates to serve as an ultraflat substrate that can possibly be functionalized. TERS investigation of monolayers adsorbed onto gold nanoplates such as alkoxy substituted azobenzene thiol and 4-nitrothiophenol is conducted. The monolayer is probed with a silver coated AFM tip in order to obtain the largest electromagnetic field enhancement and the effect of the excitation (linearly or radially polarized) is conducted. TERS is also used to probe graphene flakes and differentiate the edges of a few-layer graphene flakes with a spatial resolution better than 20 nm. Last, TERS was used to investigate single DNA molecules deposited onto gold nanoplates. The DNA, cDNA and pure plasmid were investigated with TERS probing the distribution of nucleobases at a specific location with a spatial resolution which was, in the best conditions below 10 nm

Fluorescent silver nanoclusters

PhD ThesisFluorescent metal nanoclusters (Ag2, Ag3, Ag4, and Cu7) were synthesized by reacting aqueous silver nitrate (AgNO3) or copper nitrate (Cu(NO3)2 solution with equivalent portions of aqueous sodium borohydride (NaBH4) solution in emulsion system at room temperature. Sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT) was used to stabilize the microemulsion and 2, 2, 4-trimethyl pentane (isooctane) was the bulk (oil) phase. By confining the metal ions and reducing agents to the droplets of microemulsions, the number of atoms available to form metal clusters after reduction is controlled. This thesis is concerned with the synthesis of small, fluorescent metal clusters. Ordinarily, reduction of solutions metal salts in the presence of capping ligands leads to the formation of nanoparticles of diameter in the range of 10 – 100 nm. Such particles are already metallic, have no bandgap, and do not fluoresce, but exhibit plasmon resonances. Dynamic light scattering (DLS) measurements of all emulsions indicated a predominant droplet diameter of 100 nm and a smaller diameter peak in the distribution corresponding to reverse micelles at 5 nm. Rayleigh scattering measurements were fitted to theory with the droplet diameter (50 nm) as the sole free parameter. Two reaction concentrations consisting of 90 μM and 1 mM, with an average of 4 and 40 metal ions per droplet were investigated in detail. As well as chemical reduction, photochemical reduction of Ag (I) emulsions by UV light ( = 254 nm) was also studied. UV-Vis absorption spectra did not show plasmon resonance peaks in any of the microemulsion samples. In addition, all emulsion-synthesized NCs were fluorescent with an average emission intensity counts per second (cps) in the order of 3.0 x 105 cps. Generally, two emission bands were obtained at approximately = 300 nm and = 430 nm corresponding to optical gaps of 4.13 and 2.88 eV for Ag NCs. A third band at = 610 nm may be related to aggregation was also observed. Two emission bands were also observed for the Cu NCs at = 350 nm and = 401 nm corresponding to gaps of 3.54 and 3.09 eV. Confocal microscope images confirmed the luminescence of these MNCs, and together with the transmission electron microscopy (TEM), as well as, atomic force microscopy (AFM) demonstrated that small, roughly spherical NCs were synthesized. TEM and AFM results were in agreement for the NaBH4 and photoreduced Ag NCs with estimated diameters of 1.4 – 2.4 nm. The estimated diameter of the Cu NCs was ~1.0 – 2.4 nm. Electrospray ionisation mass spectrometry (ESI-MS) results provided more the Abstract Hector Henry Oyem ii molecular formulae of: [Ag2(H2O)H-]; [Ag4B3O5BH3.2H2O]-; [Ag3(H2O)n(OH)]- and [Ag4 (H2O)6 (OH)]- (photoreduced samples), and [Cu7B3O5.BH3.2H2O]– . Alternatively, fluorescent metal NCs were prepared by reducing metal ions bound to polyvalent single-stranded deoxyribonucleic acids (ssDNAs) ligands. The concept requires that the ligand binds both the metal ions and elemental metal atoms. Three single-stranded DNAs of length 22, 29, and 34-bases which had been previously reported to facilitate the synthesis fluorescent Ag NC were chosen for investigation: (1) 5'-TGACTAAAAACCCTTAATCCCC-3' (2) 5'-AGTCACCCCAACCTGCCCTACCACGGACT-3’ (3) 5’-GCAGGTTGGGGTGACTA AAAACCCTTAATCCCC-3'. Reduction of ssDNA-bound of Ag (I) ions disfavours aggregation in bulk solution and favours instead, the formation of small, fluorescent clusters. Two prominent emission bands were obtained with ssDNA1 in microemulsion, however one broad, but weak emission at 350 nm, in associated with a complex band comprising of four peaks with a maximum at 401 nm were observed for both the ssDNA2 and 3. However, emission spectra of the three ssDNA-Ag samples in 99 % deuterium oxide (D2O) solution was different from those of the emulsion samples, with a common band at 388 nm, followed by a very broad one at 590 and 630 nm for the DNA2 and 3 respectively. Apart from the 388 nm emission, which was generally ascribed to the ssDNA molecules, no other band was seen in the spectrum of the ssDNA1-Ag sample in D2O. The results of this study demonstrate that stable NCs of specific cluster sizes could be made by adapting the reaction conditions to confine the metal ions to small reaction volumes in microemulsions or on single-stranded DNA molecules.Federal Government of Nigeria Ministry of Education Tertiary Education Trust Fund (TETFund