Developing new materials for paper-based diagnostics using electrospun nanofibers.

The use of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs) was studied. Specific chemical features of the nanofibers were achieved by doping the base polymer, poly(lactic acid) (PLA), with poly(ethylene glycol) (PEG) and polystyrene8K-block-poly(ethylene-ran-butylene)25K-block-polyisoprene10K-Brij76 (K3-Brij76) (KB). The LFAs were assembled such that the sample flowed through the nanofiber mat via capillary action. Initial investigations focused on the sustainable spinning and assembly of different polymer structures to allow the LFA format. Here, it was found that the base polymer poly(vinyl alcohol) (PVA), which was shown to function well in microfluidic biosensors, did not work in the LFA format. In contrast, PLA-based nanofibers enabled easy assembly. Three relevant features were chosen to study nanofiber-based functionalities in the LFA format: adsorption of antibodies, quantification of results, and nonspecific binding. In particular, streptavidin-conjugated sulforhodamine B (SRB)-encapsulating liposomes were captured by anti-streptavidin antibodies adsorbed on the nanofibers. Varying the functional polymer concentration within the PLA base enabled the creation of distinct capture zones. Also, a sandwich assay for the detection of Escherichia coli O157:H7 was developed using anti-E. coli antibodies as capture and reporter species with horseradish peroxidase for signal generation. A dose-response curve for E. coli with a detection limit of 1.9 × 10(4) cells was achieved. Finally, functional polymers were used to demonstrate that nonspecific binding could be eliminated using antifouling block copolymers. The enhancement of paper-based devices using functionalized nanofibers provides the opportunity to develop a broad spectrum of sensitive and specific bioassays with significant advantages over their traditional counterparts.

Effect of accessory proteins on T4 DNA polymerase replication fidelity.

The influence of replication accessory proteins on the fidelity of T4 DNA polymerase has been examined. Steady-state kinetic measurements showed that exonuclease-deficient T4 DNA polymerase, alone or with clamp loaders gp44/gp62 and polymerase clamp gp45, displays decreased binding affinity for incorrect as compared to correct dNTPs and a deceased kcat for misinsertion as compared to correct insertion. Kinetic constants were similar with and without accessory proteins, indicating that accessory proteins had little effect on misinsertion. They also had little effect on the Km value for extension of a T.T mismatch. However, the kcat value for T.T mismatch extension was fivefold higher in the presence of the clamp loader and clamp proteins. Thus, in the absence of proofreading, these accessory proteins may promote stable misincorporation. The kinetic analysis is supported by error rate determinations during gap-filling synthesis, which require both misinsertion and mispair extension. For some mispairs, the accuracy of exonuclease-deficient polymerase alone is similar to that in the presence of clamp loader, clamp and single-stranded DNA binding protein (gp32). However, exonuclease-deficient holoenzyme complex is actually less accurate than the polymerase alone for some base substitutions. We suggest that gp45 promotes extension of mismatches by tethering the polymerase to DNA, a process that may be relevant to replication past lesions or other blocks to DNA synthesis. The error rate for one-nucleotide deletions in homopolymeric runs was similar for the polymerase with or without its accessory proteins. This implies that strand misalignment errors arise during highly processive replication. Thus, either unpaired bases can migrate through the run while the DNA polymerase is bound to the template-primer, or the DNA polymerase dissociates from the DNA to allow misalignment but remains tethered to the template through interactions with the clamp. Finally, the T4 replication accessory proteins reduced by >/=10-fold the rate at which exonuclease-deficient T4 DNA polymerase generated deletions of larger numbers of nucleotides, indicating that these proteins influence replication fidelity for other than single base mutations.

Elucidation of the metal-binding properties of the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase by lanthanide(III) luminescence spectroscopy.

BACKGROUND: The exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I has a double binding site for the two essential divalent metal ions in the presence of the nucleotide monophosphate dTMP. RESULTS: The luminescence spectroscopy observed upon binding of Eu3+ to the exonuclease active site of T4 DNA polymerase was interpreted relative to the binding of Eu3+ or Tb3+ observed with KF. Both wild-type enzymes tightly bind a single Ln3+ ion but in two isomeric forms. The single mutants of KF (D424A) and T4 (D219A) also bind a single Eu3+ ion tightly, but the alignment of the coordinating ligands is altered. The KF double mutant (D355A, E357A) exhibits a markedly altered and weakened binding site (Kd = 20-26 microM). Eu3+ serves as a competitive inhibitor of Mg2+-induced polymerase and exonuclease activity, validating its use as a probe for these active sites. CONCLUSIONS: Ln3+ luminescence spectroscopy is established as a sensitive way to determine the consequences of exonuclease binding-site mutations and to examine binding site similarities and differences among DNA polymerases from different sources. The binding sites of KF and T4 DNA polymerase are shown to be quite similar.

A cellular anti-apoptosis protein is cleaved by the HIV-1 protease.

Cleavage of non-viral proteins is rarely observed with the HIV-1 protease (HIV pr). One such cleavage event occurs with bcl-2, an important cytoprotective protein. The loss of bcl-2 has biological consequences, leading to enhanced HIV replication and programmed death of the host cell. A strategy is proposed to suppress HIV with non-cleavable mutants of bcl-2.

The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase.

The fluorescent properties and their sensitivity to the surrounding environment of the nucleotide analog 2-aminopurine (2-AP) have been well documented. In this paper we describe the use of 2-AP as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of transient intermediates along the reaction pathway that are inaccessible through traditional radioactive assays. Previous studies with Klenow fragment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., & Benkovic, S. J. (1987) Biochemistry 26, 8410-8417] have revealed the presence of a nonchemical step prior to chemistry and have identified this conformational change as the rate-limiting step of correct nucleotide incorporation. During correct incorporation, phosphodiester bond formation occurs at a rate greater than the conformational change and has not been measured. However, during misinsertion, the rate of the chemical step becomes partially rate limiting and it becomes possible to detect both steps. We have successfully decoupled the chemical and conformational change steps for nucleotide insertion by KF using the misincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational change step which features hydrogen bonding between the incoming and template bases. In addition, we have utilized these same experiments to demonstrate the existence of a similar nonchemical step in the mechanism of dNTP incorporation by bacteriophage T4 DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity.

Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.

Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4.

The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.

Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2.

Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.