Current Organic Chemistry - Volume 10, Issue 4, 2006

When single DNA molecules are stretched, the measurement of the resulting force as a function of extension has yielded new information about the physical, chemical, and biological properties of these important molecules. It has been shown that double-stranded DNA molecules undergo a force-induced melting transition at high forces. Forceextension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review our studies of DNA forceinduced melting in the presence of the classical single-stranded DNA binding protein, gene 32 protein. Bacteriophage T4 gene 32 protein (gp32) is a well studied representative of a large class of single-stranded DNA binding proteins, which are essential for the replication, recombination and repair of DNA. We have developed several new single molecule methods, which when applied to gp32, have led to significant new insights about this protein's structure-function relationships. We discuss a technique for measuring Kss, the association constant of these proteins to ssDNA, which we can determine over a large range of salt concentrations not available to bulk binding studies. In addition, we have measured the noncooperative association constants (Kds) of the weak but biologically-significant interaction with double-stranded DNA as a function of salt concentration for full-length protein and *I, a truncation of gp32 lacking the 48-residue C-terminal domain. Our results have led to a quantitative model for the salt dependence of protein binding, which we postulate to be regulated by a salt-dependent conformational change within the protein involving the C-terminal domain. With this new force spectroscopy technique, we have obtained binding rates and binding free energies for these interactions under a broad range of conditions. Our methodologies should have useful applications in many areas of DNA-protein interactions.

In spite of the controversy regarding sugar and amino acid mirror-image symmetry and nature, it has been demonstrated that an intrinsic preference for left-handedness or right-handedness would depend on weak energy responsible for stabilising L-amino acids. A weak neutral current interaction involves an enantiomeric energy difference for L-amino acids to have sufficient magnitude to break open, non-equilibrium, racemic systems' chiral symmetry. L-amino acid can form complex structural atom arrangements when building proteins to allow specific chemical and molecular interactions such as enzyme-substrate and antigen-antibody complexes. Thus, pathogens can take advantage of higher vertebrates' molecular immune system's extremely well organised molecular L-amino acid composition to establish efficient evasion mechanisms. Plasmodium falciparum (the most lethal form of malaria) clearly employs its protein ligands' non-polymorphic regions when binding to specific receptors on its target cells. These sequences are normally poorly immunogenic and nonprotection inducing when used as immunogens. It has been shown that these ligands' native L-amino acid composition and their secondary structure play a vital role in maintaining a code of silence to avoid a host immune response. Our institute has established two strategies for overcoming this problem; one consists of replacing critical ligand-derived peptide binding residues by others having similar side chain mass but opposite polarity and the other consists of altering the peptide bond and the nature of α-carbon asymmetry. This review summarises the most widely used pseudopeptide approaches for novel immunogen synthesis, emphasising their potential in peptide-based vaccines and as therapeutical agents for infectious diseases such as malaria.

Iron (III) chloride is extensively used in organic synthesis as an ideal Lewis acid since it is an inexpensive, efficient, stable, environmentally friendly and a convenient agent for several useful reactions such as; polymerisations, oxidations, oxidative couplings, reductions, C C bond formation, Ferrier rearrangement, one-pot multicomponent condensations, Friedel-Crafts reactions, cyclisations, glycosidation, Prins-type cyclisation, deprotection of various functional groups, and as a reagent in key steps of natural products synthesis. This comprehensive review attempts to cover the advances in this field, which have occurred in the last five years.

Spectroscopic probes may be defined as the molecules that can react with analytes (targets) accompanying the changes of their spectroscopic (chromogenic, fluorescent, or chemiluminescent) properties; based on such changes the targets can thus be determined. Spectroscopic probes have been extensively investigated and used widely in many fields because of their powerful ability to improve analytical sensitivity and to offer greater temporal and spatial sampling capability. In this review, special interest is devoted to a new type of spectroscopic probe that is constructed with a cleavable active bond as a linker. This type of spectroscopic probe, developed greatly in the past few years, has opened a novel alternate route to the specific determination of analytes with high hydrolytic reactivity, e.g., from metal ions to enzyme activity, and enabled many biological processes to be monitored in situ and in real-time. Theoretically, various photophysical processes, such as photoinduced electron transfer, photoinduced proton transfer, and fluorescence resonance energy transfer, can be used to design spectroscopic probes with cleavable active bonds for selective detection of analytes. Herein we review the progress and application of this type of spectroscopic probe, including spectroscopic response mechanism and the probes with active bonds cleavable by enzyme, metal ion and reactive oxygen species.

Fluorescent oligonucleotides (FONs) are used in a wide variety of areas such as molecular and mechanistics biological studies, molecular diagnostics, therapeutic development, biotechnology and nanotechnology. Ever since the post-genome era, there has been an ever-increasing demand for more rapid and accurate nucleic acid detection and quantification methods. Genetic information analyses require highly sensitive and specific detection of certain sequences, single nucleotide changes, specific structures and varied reactions in different formats in vitro, in living cells and, ultimately in animals and in human beings. Ideally, a unique event could be detected and quantified using the genomic information without amplification of the nucleic acids to be analyzed. Recent developments enabling detection at the single-molecule (SM) level have opened new perspectives for applications. This review reports on the development and applications of different families of FON probes. Specific insight is given to those leading to an important fluorescent signal change upon hybridization with their targets. Applications of FON probes include real time polymerase chain reaction (PCR) quantification, detection of single-nucleotide polymorphisms (SNP), fluorescence in situ hybridization (FISH) including detection of specific messenger RNAs in living cells, analysis of gene expression, analysis of nucleic acid structures and reactions, and in nanotechnology, the assessment of molecular machine motion and functioning.