High affinity extremes in combinatorial libraries and repertoires.

By generating a large diversity of molecules, the immune system selects antibodies that bind antigens. Sharing the same approach, combinatorial biotechnologies use a large library of compounds to screen for molecules of high affinity to a given target. Understanding the properties of the best binders in the pool aids the design of the library. In particular, how does the maximum affinity increase with the size of the library or repertoire? We consider two alternative models to examine the properties of extreme affinities. In the first model, affinities are distributed lognormally, while in the second, affinities are determined by the number of matches to a target sequence. The second model more explicitly models nucleic acids (DNA or RNA) and proteins such as antibodies. Using extreme value theory we show that the logarithm of the mean of the highest affinity in a combinatorial library grows linearly with the square root of the log of the library size. When there is an upper bound to affinity, this "absolute maximum" is also approached approximately linearly with root log library size, reaching the upper limit abruptly. The design of libraries may benefit from considering how this plateau is reached as the library size is increased.

Proximity extension of circular DNA aptamers with real-time protein detection.

Multivalent circular aptamers or 'captamers' have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the 'proximity extension' assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer-thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

Plasminogen activator inhibitor-2 is highly tolerant to P8 residue substitution--implications for serpin mechanistic model and prediction of nsSNP activities.

The serine protease inhibitor (serpin) superfamily is involved in a wide range of cellular processes including fibrinolysis, angiogenesis, apoptosis, inflammation, metastasis and viral pathogenesis. Here, we investigate the unique mousetrap inhibition mechanism of serpins through saturation mutagenesis of the P8 residue for a typical family member, plasminogen activator inhibitor-2 (PAI-2). A number of studies have proposed an important role for the P8 residue in the efficient insertion and stabilisation of the cleaved reactive centre loop (RCL), which is a key event in the serpin inhibitory mechanism. The importance of this residue for inhibition of the PAI-2 protease target urinary plasminogen activator (urokinase, uPA) is confirmed, although a high degree of tolerance to P8 substitution is observed. Out of 19 possible PAI-2 P8 mutants, 16 display inhibitory activities within an order of magnitude of the wild-type P8 Thr species. Crystal structures of complexes between PAI-2 and RCL-mimicking peptides with P8 Met or Asp mutations are determined, and structural comparison with the wild-type complex substantiates the ability of the S8 pocket to accommodate disparate side-chains. These data indicate that the identity of the P8 residue is not a determinant of efficient RCL insertion, and provide further evidence for functional plasticity of key residues within enzyme structures. Poor correlation of observed PAI-2 P8 mutant activities with a range of physicochemical, evolutionary and thermodynamic predictive indices highlights the practical limitations of existing approaches to predicting the molecular phenotype of protein variants.

Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA.

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2'-deoxyuridine 5'-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E(1/2) values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with T(m) = 71 versus 75 degrees C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.

Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays.

Model single base extension (SBE) genotyping reactions with individual deoxy-, dideoxy- and acyclonucleoside triphosphates are monitored by MALDI-TOF mass spectrometry. Three non-proofreading DNA polymerases display remarkably high misincorporation (up to 64% of correct incorporation) when extending primers with single substrates at saturating concentrations. Introduction of one phosphorothioate (PS) linkage into the primer 3' terminus reduces misincorporation by these enzymes an average 1.4-fold (range 0- to 3.5-fold) versus correct incorporation. Combined use of 3'-PS primers with strongly proofreading DNA polymerases yields order of magnitude improvements in SBE fidelity over those produced by the equivalent non-proofreading enzymes. Errors are reduced to below MALDI-TOF detectable levels in almost all cases. The Sp diastereomer of the 3'-PS primer, which can be prepared in situ by incubation with proofreading polymerase, is stable to 3'-exonuclease activity over periods longer than 16 h. Products of correct extension by T7 DNAP are retained over 30-60 min during idling turnover at a dNTP concentration of 2.5 micro M, indicating that the assay can be applied over a broad range of substrate concentrations. These results suggest that the use of PS primers and proofreading polymerases will offer a simple and cost-effective means to improve fidelity in a range of single-substrate SBE assay formats.

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays.

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3'-->5' exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.

Proofreading genotyping assays and electrochemical detection of SNPs.

The use of proofreading DNA polymerases in genotyping assays offers the prospect of improved performance. To this end, we have recently used compatible DNA polymerases, protected primers, and substrates to implement proofreading single base extension (P-SBE) and proofreading allele-specific extension (PRASE) assays. Key aspects of the P-SBE and related proofreading assay formats are described here. For transduction of genotyping reactions into physical signals, electrochemical SBE implementations may offer simple, inexpensive assays in electrode array or electrophoretic formats. We have developed electrochemically-labeled nucleotides and electrode detection methods with a view to these applications. Detection of electrochemically-labeled SBE products on an oligonucleotide-modified gold electrode surface is demonstrated.

Alternate mRNA splicing in multiple human tryptase genes is predicted to regulate tetramer formation.

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.

Multitasking by multivalent circular DNA aptamers.

Nucleic acid aptamers are finding increasing applications in biology, especially as therapeutic candidates and diagnostic components. An important characteristic in meeting the needs of these applications is improved stability in physiological fluids, which is most often accomplished with chemical modification or unnatural nucleotides. In an alternative approach we have specified the design of a multivalent circular DNA aptamer topology that encompasses a number of properties relevant to nucleic acid therapeutic candidates, especially the ability to multitask by combining different activities together within a modular structure. Improved stability in blood products, greater conformational stability, antidoting by complementary circular antiaptamers, heterovalency, transcription factor decoy activity and minimal unintended effects upon the cellular innate immune response are desirable properties that are described here. Multitasking by circular DNA aptamers could similarly find applications in diagnostics and biomaterials, where the combination of interchangeable modules might generate new functions, such as anticoagulation coupled with reversible cell capture as, described here. These results provide a platform for further exploration of multivalent circular aptamer properties, especially in novel combinations of nucleic acid therapeutic modes.

Construction, stability, and activity of multivalent circular anticoagulant aptamers.

Here we describe the design and construction of multivalent circular DNA aptamers. Four aptameric binding motifs directed at blood-borne targets are used as a model set from which larger, multidomain aptamers are constructed in a straightforward manner. Intra- or intermolecular ligation of precursor oligonucleotides provides a stabilizing mechanism against degradation by the predominant exonuclease activity of blood products without the need for post-selection chemical modification. In many cases, circular DNA aptamer half-lives are extended beyond 10 h in serum and plasma, making such constructs viable for therapeutic and diagnostic applications. Duplexes and three-way junctions are used as scaffold architectures around which two, three, or four aptameric motifs can be arranged in a structurally defined manner, giving rise to improved binding characteristics through stability and avidity gains. Circular aptamers targeted against thrombin display improved anticoagulant potency with EC50 values 2-3-fold better than those of the canonical GS-522 thrombin DNA aptamer. Intrinsic duplex regions provide an opportunity to incorporate additional transcription factor binding motifs, whereas ancillary loops can be used to provide further functionality. These anticoagulant aptamers provide a starting point for merging the principles of DNA nanotechnology with aptameric functions.

Multipotential electrochemical detection of primer extension reactions on DNA self-assembled monolayers.

Electroactive nucleoside triphosphates ("electrotides") have been incorporated into primers by DNA polymerase and detected on oligonucleotide surface-assembled monolayers. Four electrotides bearing three different electroactive moieties-ferrocene, vinylferrocene, and anthraquinone-are detected in four alternative formats.

Delta tryptase is expressed in multiple human tissues, and a recombinant form has proteolytic activity.

Tryptases are neutral serine proteases selectively expressed in mast cells and have been implicated in the development of a number of inflammatory diseases including asthma. It has recently been established that the number of genes encoding human mast cell tryptases is much larger than originally believed, but it is not clear how many of these genes are expressed. A recent report suggested that the transcript for at least one of these genes, originally named mMCP-7-like tryptase, is not expressed. To further address this question, we screened tissue-specific RNA samples by RT-PCR, using primers designed to match the putative exonic sequence of this gene. We successfully generated and cloned the correctly sized RT-PCR product from mRNA isolated from the human mast cell-I cell line. Two distinct clones were identified whose nucleotide sequence matched the published sequence of the mMCP-7-like I and mMCP-7-like II genes. Transcripts were detected in a wide variety of human tissues including lung, heart, stomach, spleen, skin, and colon. A polyclonal antipeptide Ab that specifically recognizes the translated product of this transcript was used to demonstrate its expression in mast cells that reside in the colon, lung, and inflamed synovium. A recombinant form of this protein expressed in bacterial cells was able to cleave a synthetic trypsin-sensitive substrate, D-Ile-Phe-Lys pNA. These results suggest that the range of functional tryptases is larger than previously recognized. For simplicity, we suggest that the gene, transcripts, and corresponding protein product be named delta tryptase.