The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T m ‫؍‬ 107.4 ؎ 0.3°C) than avidin (T m ‫؍‬ 83.5 ؎ 0.1°C) or bacterial streptavidin (T m ‫؍‬ 75.5°C). AVR4/5 also exhibits a high affinity for biotin (K d Ϸ 3.6 ؋ 10 ؊14 M) comparable to that of avidin and streptavidin (K d Ϸ 10 ؊15 M). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed.

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Nucleic acid diagnostics is a growing sector in life sciences. Numerous applications based on the amplification of nucleic acids are utilized to determine the presence, composition, quality, or quantity of the sequence in question. However, amplification is a time-consuming, laborious, and error-prone process. Therefore, it would be preferable to perform tests with a minimized number of amplification cycles or, alternatively, completely without the amplification. We present two very sensitive sandwich assay concepts for a measurement of nucleic acids. The assays are performed either in standard microtitration wells or on microparticles coated with capture probes. Detection-probe-coated europium(III) nanoparticle labels are used for signal generation. The detection limits of the microtitration well and microparticle applications were 4.0 × 105 and 6.1 × 104 copies of target sequence, respectively. The reference assay, based on the detection of europium(III) chelate-labelled detection probes, had a detection limit of 8.5 × 107 copies. Thus, 100–1000-fold improvement in the sensitivity was achieved. The high sensitivity of the designed assays is based on the long-lifetime fluorescence of nanoparticle labels, on time-resolved fluorometry, and on signal rather than target amplification. The presented sandwich assays provide the tool for a development of direct, amplification-free detection of nucleic acids.

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.

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