Capillary circular dichroism

Circular dichroism (CD) has become an increasingly important tool in the study of biological molecules as it enables structural information to be obtained nondestructively on solution-phase samples. However, sample requirements for CD are often seen as being too high with protein backbone measurements in standard cuvettes typically requiring ～100-300 μL of 0.1 mg/ml protein. To address this issue, we have designed a new form of CD sample holder, which reduces the sample requirements of the technique by two orders of magnitude, with a sample requirement of less than 3 μl. This sample saving has been achieved through the use of extruded quartz capillaries, the sample being held within the internal diameter of the quartz capillary through capillary action. The extruded quartz capillaries exhibit remarkably little birefringence, although still transmitting high energy UV circularly polarized light. The optics associated with capillaries were investigated. A configuration has been adopted with the light beam of the spectrophotometer being focused in front of the front face of the capillary using a biconvex lens and advantage being taken of the additional focusing effect of the capillary itself. The focusing is vital to the low wavelength performance of the cell, where we have acquired reliable data down to 180 nm using a Jasco J-815 spectrophotometer. The system performance was validated with Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid), concanavalin A, lysozyme, and progesterone.     

A circular dichroism microspectrophotometer

A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.     

Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.     

Tactics targeting circular mRNA biosynthesis

Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self-splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20-bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.     

Topologically non-linked circular duplex DNA

The discovery of circular DNA, over 30 years ago, introduced an element of uneasiness in what had been, up to that point, the almost picture-perfect story of the elucidation of the molecular biology of heredity. If DNA indeed has the Watson-Crick right-handed helical secondary structure, then in circular DNA, thousands, or perhaps even millions of twists must be removed in each generation, and re-wound in the next generation. Although enzyme systems adequate for this task have long since been found and characterized, there have nevertheless arisen a number of proposals for alternative DNA structures in which the strands are topologically non-linked, so that they might separate during replication without having to be unwound. These structures have generally been put forth as theory only, and have been largely unaccompanied by experimental evidence to support their applicability to native DNA from living systems. Recently, however, a report has emerged suggesting that it might be possible to separate, intact, the individual single-stranded circular half-chromosomes which constitute the double-stranded circular chromosomes of certain plasmids. This would not be possible unless the chromosomes had one of the alternative, topologically non-linked structures. It is widely believed that after a half-century of worldwide DNA research, any significant change to the Watson-Crick structure is unlikely to stand up to scrutiny. Nevertheless, the present author has found that in many instances in which the behavior of circular duplex DNA is considered to be explicable only in terms of the topologically linked helical model, it is also possible to explain that same behavior in terms of a topologically non-linked model. It is necessary, in these instances, to make certain logical assumptions which cannot be conclusively proven at the present time. The author herein offers an example of one such instance, namely an examination of the behavior of circular duplex DNA in an alkaline titration experiment, where conformational changes in DNA are deduced from changes in its buoyant density at pH’s between 7 and 14. These data have been explained in terms of topological linkage between the DNA strands, but they can also be explained without invoking any such topological linkage, provided that the above-mentioned logical assumptions can be accepted. The principles which emerge from this are applicable to other settings in which knowledge of the topology of DNA is critical to the understanding of observed phenomena.     

Chemical synthesis of circular proteins

Circular proteins, once thought to be rare, are now commonly found in plants. Their chemical synthesis, once thought to be difficult, is now readily achievable. The enabling methodology is largely due to the advances in entropic chemical ligation to overcome the entropy barrier in coupling the N- and C-terminal ends of large peptide segments for either intermolecular ligation or intramolecular ligation in end-to-end cyclization. Key elements of an entropic chemical ligation consist of a chemoselective capture step merging the N and C termini as a covalently linked O/S-ester intermediate to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide. Many ligation methods exploit the supernucleophilicity of a thiol side chain at the N terminus for the capture reaction, which makes cysteine-rich peptides ideal candidates for the entropy-driven macrocyclization. Advances in desulfurization and modification of the thiol-containing amino acids at the ligation sites to other amino acids add extra dimensions to the entropy-driven ligation methods. This minireview describes recent advances of entropy-driven ligation to prepare circular proteins with or without a cysteinyl side chain.     

Biological synthesis of circular polypeptides

Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.     

Heparin-induced circular dichroism of chloroquine

Circular dichroism (CD) and UV/Visible absorption (UV/Vis) spectroscopy techniques were used to investigate the interaction between heparin and chloroquine, an antimalarial drug that has shown potential as an anti-prion agent. CD spectra of rac-chloroquine upon addition of heparin provide evidence of glycosaminoglycan (GAG) binding, support recent findings suggesting that interactions between heparin and antimalarial drugs are largely due to electrostatic interactions, and represent the first reported GAG-induced CD signal of a bicyclic, aromatic compound. The association constant (∼10³ M⁻¹) between chloroquine and heparin was calculated from a UV titration curve and provided additional insight into the nature of the association between these two compounds.     

环状细菌素研究进展

细菌素是一类由细菌核糖体合成的抗菌肽,是产生菌获得生存优势的重要手段。与大多数线性细菌素不同,环状细菌素具有N端和C端共价连接的特殊结构。这种环状结构赋予环状细菌素良好的耐热性、广泛的pH适应性和抗蛋白酶降解能力,在食品防腐和对治耐药性细菌领域表现出巨大的应用潜能。通过对已发现的环状细菌素结构分析发现,相对于一级结构,其三级结构的相似性更高,可以作为环状细菌素归类的依据。环状细菌素的生物合成机制尚不清楚,但其环化机制是最具价值的研究热点,可为其他一些肽类物质的合成提供支架,从而提高应用潜能。环状细菌素抑菌机制主要是在目标菌株的细胞膜上穿孔,使胞内物质外流,进而导致目标细菌死亡。其有类似于抗生素的抑菌活性和有别于抗生素的抑菌机制,为治疗日益严重的耐药性病原菌提供了可靠备选资源。本文综述了环状细菌素的构效关系、生物合成和抑菌机制方面的研究进展,希望能够对环状细菌素的深入研究和应用提供有价值的参考。     

A novel replicating circular DNAzyme

10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz₇ and C-Dz₄₈₂, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k_cat/K_M) of 7.82 × 10⁶ and 1.36 × 10⁷ M^–1·min^–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.