Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T_m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T_m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T_m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.     

Membrane lateral phase separation induced by proteins of the prothrombinase complex

Blood coagulation factors X and V, as well as prothrombin fragment 1 caused changes in the observed transition temperature (T_m) of appropriately constituted phospholipid vesicles upon binding to the membrane surface. Factor X- and prothrombin fragment 1-induced T_m shifts were calcium-dependent, while factor V changed the T_m in a calcium-independent manner. The results were consistent with clustering of the acidic phospholipid molecules due to protein binding. In all cases, protein binding to acidic phospholipid-containing vesicles caused the observed T_m to approach that for the neutral phospholipid. This resulted in a T_m increase for phospholipid mixtures containing bovine brain phosphatidylserine (PS) plus dipalmitoylphosphatidylcholine (DPPC) and a T_m decrease for mixtures of dipalmitoylphosphatidic acid (DPPA) and dimyristoylphosphatidylcholine (DMPC). Maximum T_m shifts induced in PS-DPPC (10:90) vesicles were very similar for all the prothrombinase proteins and the extent of the change was proportional to the actual amount of membrane-bound protein as determined by light-scattering techniques. For the vitamin K-dependent proteins, T_m changes were greater in the presence of protein plus calcium than in the presence of calcium alone, indicating that lateral phase separation occurs subsequent to initial protein-membrane contact. Lateral phase separation of acidic phospholipids appears to be an important process in the formation of the prothrombinase complex.     

Kinetic screening of antibody-Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V_NAR single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors.     

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3′-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T_m) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5′-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T_m (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T_m and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.     

Melting temperature of surface-tethered DNA

A method for the accurate determination of the melting temperature (T_m) of surface-immobilized DNA duplexes that exploits the fluorescence-quenching properties of gold is reported. A thiolated single-stranded DNA probe is chemisorbed onto a gold surface and then hybridized to a fluorophore-labeled complementary sequence. On formation of the duplex, the fluorescence of the label is effectively quenched by the gold surface. As the temperature is increased and the duplex denatures, the fluorophore label moves away from the gold surface and the fluorescence signal is again observed. The increase in fluorescence is measured as the temperature is ramped, and using first-derivative plots, the T_m is determined. To demonstrate the approach, the T_m of the cystic fibrosis DF508 mutation was determined in three different phases: in solution, in suspension immobilized on gold nanoparticles, and immobilized on gold film-coated substrate. The technique was further applied to optimize conditions for differentiation between a surface-immobilized DF508 mutant probe and a mutant/wild-type target exploiting increasing stringency in varying salt and formamide concentrations. The approach has application in optimization of assay conditions for biosensors that use gold substrates as well as in melting curve analysis.     

Effect of selenite and selenate on rat liver nuclear 3,5,3′-triiodothyronine (T3) receptor

The present study was undertaken to investigate the effects of selenite (Se^IV) or selenate (Se^VI) on nuclear T₃ receptors of rat liver. Selenite at 0.1 μM (p<0.01) inhibited the T₃ specific binding to rat liver nuclear receptors. The specific binding of the T₃ receptor was fully restored when even 1.0 μM selenite was separated from the T₃ receptor by gel filtration. No inhibitory effect of selenite (up to 100 μM) on the T₃ binding to nuclear receptor was found in the presence of 1.0 mM dithiothreitol. The rate of dissociation of the T₃-nuclear receptor complex was effectively increased by 0.1 μM selenite. Selenate up to 1 mM as well as sulfite or sulfate up to 0.1 mM did not exert an inhibitory effect on T₃ receptors. The results based on the in vitro experiments suggest that the selenium in the form of selenite may reversibly affect the T₃ binding on the receptor molecule.     

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen® screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K_m values were determined as 5.6 μm for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1× and 10× K_m concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.     

Ligand binding analysis and screening by chemical denaturation shift

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (T_m shift) has become a popular approach to identify potential ligands. However, T_m shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (?60 °C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples.     

A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as K_D, k_on, k_off, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand–p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in T_m values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.     

The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (K_i = 0.29 ± 0.07 μM, ΔT_m = 8.5 °C). This compound’s effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.     

Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (T_m) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the T_m of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a T_m around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9–DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.     

Probing the Combined Effect of Flunitrazepam and Lidocaine on the Stability and Organization of Bilayer Lipid Membranes. A Differential Scanning Calorimetry and Dynamic Light Scattering Study

Combined effects of flunitrazepam (FNZ) and lidocaine (LDC) were studied on the thermotropic equilibrium of dipalmitoyl phosphatidylcholine (dpPC) bilayers. This adds a thermodynamic dimension to previously reported geometric analysis in the erythrocyte model. LDC decreased the enthalpy and temperature for dpPC pre- and main-transitions (ΔH _p, ΔH _m, T _p, T _m) and decreased the cooperativity of the main-transition (ΔT _1/2,_m). FNZ decreased ΔH _m and, at least up to 59 μM, also decreased ΔH _p. In conjunction with LDC, FNZ induced a recovery of ?T _1/2,m control values and increased ΔH _m even above the control level. The deconvolution of the main-transition peak at high LDC concentrations revealed three components possibly represented by: a self-segregated fraction of pure dpPC, a dpPC–LDC mixture and a phase with a lipid structure of intermediate stability associated with LDC self-aggregation within the lipid phase. Some LDC effects on thermodynamic parameters were reverted at proper LDC/FNZ molar ratios, suggesting that FNZ restricts the maximal availability of the LDC partitioned into the lipid phase. Thus, beyond its complexity, the lipid–LDC mixture can be rationalized as an equilibrium of coexisting phases which gains homogeneity in the presence of FNZ. This work stresses the relevance of nonspecific drug–membrane binding on LDC–FNZ pharmacological interactions and would have pharmaceutical applications in liposomal multidrug-delivery.     

Assessing compound binding to the Eg5 motor domain using a thermal shift assay

Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5′-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP-Eg5 complexes using a thermal shift assay. We measured up to a 7 °C increase in the thermal melting (T_m) of Eg5 for an inhibitor that produced IC₅₀ values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP-Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (μM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.     

A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus α-amylase—the effect of calcium ions

The thermal stability of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This α-amylase is homologous to other Bacillus α-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T_m of approximately 89 °C and DSC thermograms can be described using a one-step irreversible model. A 5 °C increase in T_m in the presence of 10-fold excess CaCl₂ was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30–40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[β-aminoethyl ether] N,N,N′,N′-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 °C lower T_m as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous α-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.     

Reexamination of the interaction between ferredoxin:NADP reductase and NADP

A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave K_d values near 10 μm which are consistent with recently determined values for K_m,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (K_d approx 1 mm) was also detected.     

Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T_m), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T_m, which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T_m lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T_m phosphatidylcholines, cholesterol, and high-T_m lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T_m lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T_m lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T_m lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T_m lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T_m lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T_m lipids can stabilize ordered domain formation by high-T_m lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T_m and high-T_m lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

Calorimetric studies of bovine rod outer segment disk membranes support a monomeric unit for both rhodopsin and opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (T_m) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T_m of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T_ms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T_m remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T_m of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.