The schematic representations of HIV-1 Gag domains and assembly process.

(A) Gag domains and key basic residues or basic residue clusters discussed in this review. Upon cleavage of Pr55Gag, individual domains give rise to mature Gag proteins, MA, CA, p2 (SP1), NC, p1 (SP2), and p6. NTD, N-terminal domain; CTD, C-terminal domain; ZF, zinc finger. (B) Interactions between basic residues in Gag and polyanions play important roles at different steps of virus particle formation and post-assembly processes. In some cases, the basic residues in Gag and the polyanions switch their interaction partners through the transitions from one step to another. PI(4,5)P2, phosphatidylinositol (4,5) bisphosphate; IP6, inositol hexakisphosphate; vRNA, viral genomic RNA.</p

Design, Synthesis, and Cellular Uptake of Oligonucleotides Bearing Glutathione-Labile Protecting Groups

Glutathione-labile protecting groups for phosphodiester moieties in oligonucleotides were designed, synthesized, and incorporated into oligonucleotides. The protecting groups on the phosphodiester moieties were cleaved in a buffer containing 10 mM glutathione, which was used as a model of intracellular fluid. Cellular uptake of oligonucleotides bearing glutathione-labile protecting groups was strongly affected by the location and number of the protecting groups

Conjugatable and Bioreduction Cleavable Linker for the 5′-Functionalization of Oligonucleotides

An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5′-terminal ends of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and incorporated at the 5′-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides

The NMR Structure of a DNA Dodecamer in an Aqueous Dilute Liquid Crystalline Phase

The solution structure of the DNA dodecamer d(CGCGAATTCGCG)2 has been studied in an aqueous liquid crystalline medium containing 5% w/v bicelles. These phospholipid particles impose a small degree of orientation on the DNA duplex molecules with respect to the magnetic field and permit the measurement of dipolar interactions. Experiments were carried out on several samples with different isotopic labeling patterns, including two complementary samples, in which half of the nucleotides were uniformly enriched with 13C and deuterated at the H2‘ ‘and H5‘ positions. From this, 198 13C−1H and 10 15N−1H one-bond dipolar coupling restraints were derived, in addition to 200 approximate 1H−1H dipolar coupling and 162 structurally meaningful NOE restraints. Although loose empirical restraints for the phosphodiester backbone torsion angles were essential for obtaining structures that satisfy all experimental data, they do not contribute to the energetic penalty function of the final minimized structures. Except for additional regular Watson−Crick hydrogen bond restraints and standard van der Waals and electrostatic terms used in the molecular dynamics-based structure calculation, the structure is determined primarily by the dipolar couplings. The final structure is highly regular, without any significant bending or kinks, and with C2‘-endo/C1‘-exo sugar puckers corresponding to regular B-form DNA. Most local parameters, including sugar puckers, glycosyl torsion angles, and propeller twists, are also tightly determined by the NMR data. The precision of the determined structures is limited primarily by the uncertainty in the exact magnitude and rhombicity of the alignment tensor. This causes considerable spread in parameters such as the degree of base-pair opening and the width of the minor groove, which are relatively sensitive to the alignment tensor values used

¹⁵N−¹⁵N <i>J</i>-Coupling Across Hg^II: Direct Observation of Hg^II-Mediated T−T Base Pairs in a DNA Duplex

N−N J-coupling across a metal center (2JNN) was clearly detected in a biological macromolecule (DNA duplex) for the first time. By using 2JNN, the base pairing mode of mercury-mediated T−T pairs (T−HgII−T) was definitely determined. This pairing mode was found to be a novel metal ion-binding mode for DNA and RNA molecules, in which imino proton−metal exchange processes are included. Accordingly, 2JNN is highly important for the determination of the chemical structures of metal-mediated base pairs

Synthesis and Characterization of Cell-Permeable Oligonucleotides Bearing Reduction-Activated Protecting Groups on the Internucleotide Linkages

Cell-permeable oligodeoxyribonucleotides (ODNs) bearing reduction-activated protecting groups were synthesized as oligonucleotide pro-drugs. Although these oligonucleotides were amenable to solid-phase DNA synthesis and purification, the protecting group on their phosphodiester moiety could be readily cleaved by nitroreductase and NADH. Moreover, these compounds exhibited good nuclease resistance against 3′-exonuclease and endonuclease and good stability in human serum. Fluorescein-labeled ODNs modified with reduction-activated protecting groups showed better cellular uptake compared with that of naked ODNs

Well-Controlled Polymerization of Phenylacetylenes with Organorhodium(I) Complexes: Mechanism and Structure of the Polyenes

A tetracoordinate rhodium complex, Rh(C⋮CC6H5)(nbd)[P(C6H5)3] (nbd = 2,5-norbornadiene), combined with 4-(dimethylamino)pyridine (DMAP) is an excellent initiator for the stereospecific living polymerization of phenylacetylene and its m- and p-substituted derivatives. The rhodium initiator can be generated efficiently by dissociation of triphenylphosphine from isolable Rh(C⋮CC6H5)(nbd)[P(C6H5)3]2 or by reacting Rh(CH3)(nbd)[P(C6H5)3]2 or [Rh(OCH3)(nbd)]2/P(C6H5)3 with one equivalent of phenylacetylene. The use of a phenylethynyl group, triphenylphosphine, and NBD ligand attached to the rhodium center is crucial for the well-controlled polymerization of phenylacetylenes. An additive, DMAP, is necessary to attain low polydispersities of the polymer products. An active rhodium(I) complex bearing a growing polymer chain, NBD, and P(C6H5)3 was isolated from a reaction mixture and was characterized by NMR, GC−MS, XPS, and elemental analyses. The isolated active polymer initiates the further polymerization of the same monomer or substituted ones with an almost 100% initiation efficiency to give higher molecular weight homopolymers or block copolymers, respectively. Detailed NMR structural analysis of the products indicated that the polymerization with the rhodium(I) complexes proceeds via a 2,1-insertion mechanism to provide stereoregular poly(phenylacetylene)s with cis−transoidal backbone structure

Optimized Method for Computing ¹⁸O/¹⁶O Ratios of Differentially Stable-Isotope Labeled Peptides in the Context of Postdigestion ¹⁸O Exchange/Labeling

Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs have made trypsin-catalyzed 18O postdigestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme−substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin-catalyzed 18O postdigestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both accuracy and precision are improved utilizing this rigorous mathematical approach. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its nonmyristylated mutant

MR image before and after shunt surgery.

(A) MR image shows linear enhancement along the cerebellar folia. (B) FLAIR image obtained prior to VP shunt shows enlarged lateral ventricles with periventricular high intensity. (C) FLAIR image after VP shunt shows decreased size of the lateral ventricles and improved periventricular high intensity three months after shunting.</p

Optimized Method for Computing ¹⁸O/¹⁶O Ratios of Differentially Stable-Isotope Labeled Peptides in the Context of Postdigestion ¹⁸O Exchange/Labeling

Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs have made trypsin-catalyzed 18O postdigestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme−substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin-catalyzed 18O postdigestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both accuracy and precision are improved utilizing this rigorous mathematical approach. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its nonmyristylated mutant