DYNAMICBIOSENSORS. Chip functionalization DNA-encoded addressing and chip configuration. Technology Information #120

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1 Technology Information #120 Chip functionalization DNA-encoded addressing and chip configuration The switchsense biochip features 24 detection spots, which are arranged in 2 or 4 seperate flow channels. The microelectrodes are individually functionalized with capture molecules through DNA encoded addressing. Before a switchsense chip can be exposed to target molecules of interest, the chip surface needs to be functionalized with one of the interaction partners. A versatile and parallel strategy employing DNA-DNA recognition is used for coupling capture molecules to the electrically actuated nanolevers. The microlectrodes on a switchsense chip come prefunctionalized with single-stranded DNA. One end of the oligonucleotides is covalently tethered to the surface via gold-sulfur bonds, while the opposing distal end features a fluorescent dye for optical detection. After hybridization with their complementary sequences (cdna), the DNAs become double-stranded helices ideal rod-like nanolevers for efficient switching. Functionalization methods The complementary strand is modified with a reactive group for conjugating capture molecules (small molecules or proteins) to it. A number of modifiers and bifunctional cross-linkers are available for addressing a wide variety of different chemical groups on the capture molecule. Reactive group on capture molecule Cross linker Modifier on cdna Amino NHS Amino NHS X maleimid Thiol Amino NHS X azide Alkyne Thiol Maleimid Thiol maleimid X NHS Amino Thiol maleimid X azide Alkyne Alkyne Azide Alkyne azide X maleimid Thiol Alkyne azide X NHS Amino His-tag Ni 2+ Tris-NTA GST-tag Glutathione Biotin Streptavidin Strep-tag Streptavidin Streptavidin Biotin Dig-binding Digoxigenin Fc part of IgG Protein A Fc part of IgG Protein G Fab part of IgG Protein L X different types of linkers and lengths are available Figure 1 Bio-layer architecture of the chip. The standard DNA nanolever has 48bp and is 16 nm long. 1

2 Two alternative strategies can be used for functionalizing the nanolevers with capture molecules: (i) on-chip conjugation or (ii) in-vitro conjugation, cf. Figures 2 and 3. During the on-chip conjugation method, the reaction between the capture molecule and the cdna strand takes place in the flow channel whilst the chip is installed in the switchsense instrument. The in-vitro conjugation is carried out in vials ex-situ, which is advantageous during chip regeneration as the previously prepared stock solution of the conjugate can be used to regenerate the chip in two automated steps with Figure 2 On-chip conjugation of capture molecules to double-stranded DNA. Complementary DNA with a suitable modifier for conjugation is hybridized to the pre-immobilized tethered strand (3 min). Upon incubation of the microelectrodes with capture molecules, a covalent linkage between reactive groups on the capture molecule and the modifier is established (1-10 min). The interaction between the target protein and the capture molecule is measured. The surface is regenerated by denaturing the DNA duplex, which removes the cdna strand along with the capture and target molecules (1 min). Figure 3 In-vitro conjugation of capture molecules to cdna using provided kits and protocols, followed by hybridization of cdna conjugates to the detection surface. After the interaction measurement, the surface can be regenerated by denaturing the DNA duplex, which removes the cdna-conjugate and resets the electrode to its initial single-stranded state. 2

3 exceptional reproducibility. In addition, it is more straightforward to individually address several electrodes in parallel with different capture molecules, thereby taking advantage of the DNA encoded addressing method. As an alternative to the standard 16 nm nanolever, a modular system of short anchor strands (20 bp, 6.5 nm) in conjunction with a generic fluorescent labelsequence is available. It is particularly useful for experiments with DNA-binding proteins, where a large number of DNA sequences needs to be economically tested. distance between two adjacent nanolevers is 30 nm, which provides the levers with enough space to efficiently switch their conformation (standing/lying) while keeping the density of capture molecules presented to the analyte solution favorably low. This is essential for facilitating an unhampered association and dissociation of target molecules and for obtaining accurate, artifact-free binding data. Figure 5 Low density layers for efficient switching and optimal target accessibility. Figure 4 Coupling of variable DNA sequences via anchor strands. The sequences under investigation do not need to be labeled, but are designed with overhangs so that they can be coupled to the anchorand label-strands on their opposing ends. Less is more: nanolever numbers and material consumption There are only 1 million (1.6 attomol) DNA nanolevers on a detection spot, which is an exceptionally low number. It partly results from the small area of the microelectrodes (0.01 mm²), which is comparable to the cross-section of a human hair. Importantly however, the density of nanolevers on the surface is very low (~10 10 levers/cm²), which is carefully adjusted during production of the chip and can be fine-tuned over one order of magnitude by the user through a proprietary desorption process. Hence, the average For a typical functionalization following an in-vitro conjugation, 50 µl of a 100 nm conjugate solution is used to incubate a flow channel for 3 min (step in Fig. 3). This corresponds to a consumption of 5 pmol of capture molecule, or 0.2 µg when assuming a molecular weight of 50 kda. Chip regeneration The switchsense platform uses one consistent regeneration procedure, irrespective of the types of molecules under investigation. There is no need to develop or to optimize regeneration conditions, as the capture molecules used (which might still be be occupied with targets) are simply washed away by a brief treatment with basic ph solution. This treatment denatures the base-pairing between DNA-tether and cdna, whilst leaving the covalently bound DNAtether on the surface for repeated use. The loss of signal during a regeneration cycle is less than 5%, allowing the same flow channel (80 per chip) to be used repeatedly for up to 20 times. It should be noted that, in principle, the measurement accuracy does not suffer due to repeated regeneration cycles, because 3

4 switchsense does not rely on an absolute fluorescence measurement but assesses molecular motion/dynamics instead. However, the time needed to acquire signals of comparable signal-to-noise ratios increases when the DNA layer quality declines, which in practice limits the number of regenerations possible. Getting parallel: DNA encoded addressing In a switchsense experiment, the DNA nanolevers fulfill a dual purpose: to act s negatively charged nano-rods and to use their base-pairing capabilities to direct capture molecules to their designated detection spots. This enables a parallel and selective functionalization of all microelectrodes in a flow channel simultaneously. within one flow channel can be incubated simultaneously with a mixture of the conjugates in step. The specially selected sequences ensure that the conjugates hybridize with the DNA-tethers on the designated electrodes only. The DNA sequences of the standard 48 bp DNA levers as well as the 20 bp anchor strands have been selected for minimal cross-reactivity between the different detection spots, thus unwanted hybridization reactions are practically negligible. Chip layout The number of flow channels on the chip as well as the number of different DNA-tether sequences can be chosen according to the experimental requirements. If only few interactions need to be investigated, the electrode functionalization can feature replicates in favor of better measurement statistics. If a large number of diverse interactions are to be investigated simultaneously, the chip can be configured for maximal parallelization. No. of flow channels No. of electrodes per channel No. of different DNA sequences per channel Examples for chip configurations: Figure 6 DNA encoded addressing. Capture molecules are conjugated to different, unique cdna sequences in step. Subsequently, all electrodes 4 channels with 6 electrodes, each channel is modified with 2 different sequences (4 6-2s) 2 channels with 12 electrodes, each channel is modified with 6 different sequences (2 12-6s) 4

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