The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures

Abstract

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperaturesThe biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K d , in the order of 4610 214 M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 707C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology. IntroductionThe strong interaction between avidin and biotin was discovered as early as 1941 [1]. Avidin is a protein commonly purified from chicken egg white while biotin is a vitamin found in all cells. Streptavidin, a bacterial homologous protein to avidin, isolated from the actinobacterium Streptomyces avidinii, is more frequently used than avidin and is commercially available also in a number of engineered forms. The structure of the biotin-streptavidin complex has been described by several groups [2,3], showing a b-barrel structure of streptavidin binding biotin into its interior. The binding between avidin/streptavidin and biotin has long been regarded as the strongest, noncovalent, biological interaction known, having a dissociation constant, K d , in the order of 4610 214 M [4]. The bond forms very rapidly and is stable in wide ranges of pH and temperature [1,5].The strong interaction has led to a large number of research and diagnostic applications using avidin-biotin or streptavidin-biotin technology. The strength and reliability of the interaction underlie its importance in biotechnology, but the interaction is also a model for high-affinity receptor ligand binding. In most assays, streptavidin is coupled to a solid phase, such as a magnetic bead, a microtiter plate, or a biosensor chip, while biotin is coupled to the moiety of interest, often a nucleic acid, protein, or antibody. However, harsh conditions, such as formamide treatment combined with high temperatures, have been required to separate biotin from streptavidin, resulting in not only denatured streptavidin molecules [5] but also in limitations of downstream applications due to deterioration ...

Programmed folding of DNA origami structures through single-molecule force control

“…Then, we washed the magnetic bead again with distilled water twice. The antisense strand was released from the bead by heating the sample to 63°C in 200 ml of distilled water 41 . Typical concentration of resulting single-stranded DNA was 40 nM in 200 ml.…”

Purification of Peptides in High-Complexity Arrays

“…Streptavidin, a protein from Streptomyces bacteria, binds to biotin forming one of the strongest known non-covalent bonds. [159] The DyLight 680-streptavidin conjugate (DyLight 680: λex=682 nm, λem=712 nm) could, thus, be used to additionally stain the array after spotting (Figure 39, c)). Arrays stained with DyLight 680-streptavidin were scanned using the Odyssey Infrared Imager.…”

Microheater platform for selective detachment of DNA

“…Biotinlyated DNA is then covalently linked to streptavidin. It has been reported that the streptavidin-biotin bond can be broken in an aqueous solution at temperature above 70 C. 10 Consequently, application of a spatial temperature field on DNA immobilized on glass offers a purely thermal means of spatially controlled DNA detachment. By manipulating the temperature field, it may be possible to control where on a glass chip DNA detaches and where it remains attached.…”

“…It is also well known that the biotin-streptavidin bond, which is commonly used to covalently attach DNA to glass surfaces 9 has a temperature-dependent chemistry. 10 This bond has been observed to break at around 70 C. 10 By subjecting surface-bound DNA to spatial temperature gradients, it may be possible to detach DNA from only specific locations on a microarray chip. Thermal "zones" based on an externally applied temperature field may enable selective adherence and detachment of DNA in a microarray chip.…”