Different ways of attaching a λ - DNA on a functionalized

Amine – thiol cross-linking

First of all we studied the possibilities to end-graft thiol-modified λ - DNA on amino silanized surface. From the amine – thiol reactive heterobifunctional cross-linker family we tested two possibilities: SMCC and sulfo-SIAB.

(a) SMCC (Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is not soluble in water and therefore it has to be first dissolved in dimethyl-sulfoxide (DMSO). We dissolved SMCC typically in concentration of 2 mg/ml in solution of 10 % DMSO, the rest being PBS at pH 7.2 (recommended by the manufacturer). The solution was immediately pipetted on the amino-silanized glass surface and left to react for one hour. In the presence of primary amine nucleophiles, such as the amino group of APTES, the NHS ester reacts in a nucle-ophilic attack creating a stable (covalent) amide link with the APTES molecule (1). N-hydroxysuccinimide is released in this reaction as a byproduct [19]. After one hour the cross-linker was washed away with PBS and finally with TBE.

+

Figure 4.1: The clean glass surface is first silanized with APTES. In the following step the amino moiety of the APTES is cross-linked with SMCC in a reaction where the NHS ester derivative reacts, creating a stable (covalent) amide bond with the remaining part of SMCC (1). After that thiol-modified λ - DNA is incubated with the malemide group covered surface (2). The maleimide group of SMCC forms a thioether (3) with thiol-modified λ - DNA.

The surface is now covered with maleimide groups (see Fig. 4.1), and can be let to react with thiol-modified DNA (2). One of the carbons adjacent in the maleimide group experiences nucleophilic attack by the thiol group and forms a thioether (3) [19].

We started by incubating DNA loaded with YOYO-1 with the maleimide surface in various buffer compositions. The manufacturer recommends to use PBS at pH of 6.5−7.5 when letting the thiol groups to react with maleimide groups. We observed that DNA is sticking unspecifically at the surface (see Fig. 4.2 (a)) when working at pH 7.5 and below. At higher pH the sticking is reduced but so is the number of end-grafted molecules also. When keeping the pH constant at 7.5 and varying the NaCl concentration of the buffer between 0−1.5 M the optimal reaction conditions were found to be at pH 7.5 and 0.5M NaCl in TBE buffer. Even under these conditions there was sticking directly after the incubation. When the samples were let to stay overnight in TBE pH 9.0 the previously stuck DNA had been released from the surface and the carpet seemed to be normal. The estimation of the carpet quality was done by using the microscope to judge the homogeneity and density of the DNA carpets. The SMCC chemistry was not further tested since waiting for the DNA to release from the surface would not be experimentally practicable.

(b) This led us to test sulfo-SIAB (N-Succinimidyl(4-iodoacetyl)aminobenzoate). Experimentally sulfo-SIAB is almost the same as SMCC. The main differences are that (i) instead of maleimide group sulfo-SIAB contains a iodoacetyl group that reacts with thiol-modified DNA at higher pH compared to the maleimide group and (ii) that sulfo-SIAB is water soluble.

When working with sulfo-SIAB a difficulty arises from the fact that sulfo-SIAB is sensitive to light and all the steps of the reaction have to be done in dark.

In the first step the cross-linker, in concentration of 2 mg/ml, is let to react with APTES silanized surface in PBS at pH 7.2 with 10 mM EDTA. This part of the reaction incorporates the sulfo-NHS reaction and is exactly the same as already described with SMCC (4). After the sulfo-NHS reaction the surface is covered with iodoacetyl groups (5). In the following reaction the halogen is replaced by the attacking nucleophile, i.e. the thiol group, and a stable thioether (6) is formed [19]. The iodoacetyl group according to the manufacturer works at pH values 7.5−8.5 and is most selective to thiol groups at pH 8.3. The schematic reaction pathway is presented in Fig. 4.3.

We incubated DNA in various pH values around pH 8.3 with varying NaCl concentrations and found that the DNA carpets incubated in PBS at roughly pH 8.3 with 0.5 M NaCl seemed to be qualitatively the best, meaning that when observed with microscope the carpets were homogeneous and as dense as carpet done with glutaraldehyde and streptavidin (see Fig. 4.2 (b)). Problematic in sulfo-SIAB is the relatively high price, the fact that it is photoreactive and the need to store sulfo-SIAB under argon atmosphere at −20^◦C.

Amine – biotin reactive cross-linking chemistry

Biotin-modified DNA can be end-grafted to the surface over streptavidin. Strep-tavdin has four biotin binding sites so streptavidin can be sandwiched between

(a) (b)

(c) (d)

Figure 4.2: We used microscopy to characterize the surface densities and the surface grafting quality of the λ - DNA carpets: a) Sticking of the molecules to the surface: no fluctuating molecules are seen and most of molecules are found at the surface. b)Dense carpet: it is already very hard to tell a difference between separate molecules. All the molecules seem to fluctuate but do not diffuse away.

c) Moderate density: molecules are easily detected as single molecules. d) Low density: at the surface are only few molecules to be found. All the snapshots are 30µm× 40µm.

NH₂

Figure 4.3: Sulfo-SIAB cross-linker links first the amino moiety of the amino silane with the SIAB molecule over a stable amide bond (4). In the following step the iodoacetyl coated surface is incubated with thiol-modified λ - DNA (5).

The iodoacetyl group of sulfo-SIAB reacts with thiol-modifiedλ- DNA and forms the thioether (6).

biotinylated surface and biotinylated DNA as presented in Fig. 4.4 where the sur-face biotinylation was done with succinimidyl-6-(biotinamido) hexanoate better known as EZ −Link^r.

The EZ − Link^r is soluble in PBS with 10 mM EDTA at pH 7.2. The concentration of EZ −Link^r was typically 2 mg/ml but we tested also other concentrations from 0.004 mg/ml up to 3 mg/ml. EZ−Link^r was left to react typically for one hour but also longer incubation times were tested. The first step of the reaction is the same as already described with SMCC and sulfo-SIAB: the carbonyl group of the cross-linker experiences an nucleophilic attack from the surface amine and the sulfo-NHS leaves, creating a stable amide (7).

After one hour the excess EZ − Link^r was washed away with PBS and the biotinylated surface was ready for streptavidin incubation (8). The streptavidin was incubated also for one hour at a concentration of 0.1 mg/ml, buffer being typically PBS at pH 8.The streptavidin covered surface was finally washed with PBS and TBE respectively and then incubated with biotin-modified λ - DNA (9). The concentration of the DNA was typically 2 ng/µl in TBE with 0.5 M NaCl at pH 9. Qualitatively seen all the produced DNA carpets had moderate or low density as for example can be seen in Fig 4.2 (c) and we did not succeeded to improve on this by varying the EZ−Link^rconcentration, pH of the buffer or the NaCl concentration of the buffer. Furthermore this end-grafting possibility is not covalent as already discussed in chapter 2.

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Figure 4.4: EZ−Link^r is used to biotinylate primary amine containing macro-molecules and surfaces. In the first step the sulfo - NHS is leaving and the rest of the cross-linker reacts first with the primary amino group (7) found at the surface.

Later streptavidin is sandwiched between surface biotin (8) and biotin-modified λ - DNA (9).

Carboxyl – amine cross-linking

In order to accomplish a cross-link between carboxylated surface and amine modified λ - DNA, we tested so called zero-length cross-linkers. The ”zero-length” cross-linkers mediate link between two molecules to be cross-linked form-ing a bond without addform-ing any additional atoms [19]. In our case the sur-face amino group has to be converted to a carboxyl group¹ before the zero-length cross-linkers can be used. From the zero-zero-length cross-linker family we chose to test two possibilities: 1,1´-Carbonyldiimidazole (CDI) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

(a) The reaction scheme of the CDI is shown in Fig. 4.5. The experimental protocol was following: The carboxylated glass slide was treated with CDI dis-solved in diethyl ether at a concentration of 3 mg/ml and left to react for two hours at 37^◦C. The CDI reacts with carboxyl group and forms an N-acylimidazole intermediate (10). This intermediate can then react with amino group and form stable amide bond [19].

After the reaction with CDI the glass slide was washed with ethanol and the amino-modified DNA, at a concentration of 2 ng/µl, was brought to the surface in PBS buffer at pH 8.4 containing 0.5 M NaCl (11). DNA was let to incubate roughly one hour after which the result was observed with microscope (12). Qualitatively seen DNA in fact seemed to be end-attached to the surface

1The carboxylation of the amino silanized surfaces is presented below in context with the silanization.

O

Figure 4.5: With APTES silanized slides can be carboxylated with succinic an-hydride (explained in context with silanization). The CDI reacts with carboxyl group and forms N-acylimidazole intermediate (10) which can react further with amino group containing λ - DNA (11) and form an amide (12).

but the densities were not satisfactory, for example, in comparison with carpets done with sulfo-SIAB. The biggest problem with CDI was the use of diethyl ether as a solvent. Diethyl ether evaporates very easily and makes the handling very difficult, even so that we were not able to complete any experiments without some problems caused by the evaporation of diethyl ether. Furthermore it is toxic, causing irritation, allergic reactions or in worst case even coma. In all it is such a substance that one wants to avoid when there is also other possibilities such as EDC.

(b) EDC reaction schema is presented in Fig. 4.6 where the carboxyl group terminated APTES molecule reacts with EDC (13). In the first step the O-acylisourea reacts with the carboxylate group and it forms an active intermediate which hydrolyses in seconds. However, when sulfo-NHS is added to the solution (14), a reactive ester intermediate is formed with the carboxyl group and sulfo-NHS. This intermediate stays active for hours and is sensitive to a nucleophilic attack from the amino group of the amino-modified λ - DNA (15) or from the surface amino groups of streptavidin, resulting in a stable amide [19].

We mixed 2 mg/ml of EDC and 2 mg/ml of sulfo-NHS in MES buffer at pH 5.5 and left this solution react on a carboxyl-terminated silanized glass for 20 minutes. After washing with PBS amino-modified DNA was brought to the surface in PBS buffer at pH 7.2 with 0.5 M NaCl and left to incubate for two hours. After the incubation the samples were washed with TBE and observed under the microscope. The direct binding of the λ - DNA to the EDC-activated surfaces was successful but did not result in dense carpets. Therefore we tested also the option to cross-link streptavidin directly from its surface amino groups

O

Figure 4.6: Carboxyl terminated APTES silane reacts with EDC and forms O-acylisourea intermediate (13) which can hydrolyze very rapidly. By adding sulfo-NHS simultaneously with EDC to the solution, a sulfo-sulfo-NHS ester intermediate can be formed (14) which stays active for hours (15). This intermediate can react with amino-modified λ - DNA and form an amide (16) [19].

to the EDC activated surface and so substitute glutaraldehyde as a cross-linker between surface amino group and streptavidin. This worked better than direct end-grafting of DNA but nevertheless the densities of the carpets were not on the level of the ones produced, for example, with sulfo-SIAB or with glutaraldehyde and streptavidin. Furthermore physisorption of streptavidin cannot be ruled out in this end-grafting possibility and as already mentioned before the biotin-streptavidin linkage is not covalent.

Gold – thiol coupling revisited

Gold – thiol linkage was already presented in chapter 2. The need sometimes to stretch the DNA between two gold surfaces drove us to test the possibility to thiolate streptavidin (introduce thiol groups on the surface of streptavidin).

This gives us a possibility to stretch DNA modified from one end with thiol and from other end with biotin between two gold surfaces, using streptavidin as a cross-linker, as shown in Fig. 4.7 (a). The thiolation of streptavidin is presented in Fig. 4.7 (b) where in the first step N-succinimidyl S-acetylthioacetate (SATA) reacts with the primary amino groups on the surface of streptavidin (20). The thiol groups are introduced to the end of the SATA molecule just before use.

This is done with hydroxylamine which is a strong reducing agent and is used for deacylation (21), in other words to remove an acetyl group, and so to generate a thiol group in the end of the SATA molecule.

In practice, as SATA is not soluble in water, we prepared typically a stock solution of SATA in DMSO at a concentration of 108 mg/ml. We store our streptavidin in milli-Q water in concentration of 1 mg/ml but as SATA needs special conditions to work properly the buffer must be changed. This was done by size-exclusion chromatography withZeba^{T M} spin columns in a microcentrifuge and as a new buffer PBS with 0.15 M NaCl at pH 7.2−7.5 was chosen. We took 1.5 µl of the freshly prepared SATA solution and mixed it with 150µl of streptavidin solution. This mixture was left to react for 30 minutes at room temperature.

These concentrations provide a ratio of roughly 250 : 1 molecules of SATA to streptavidin. After the reaction of SATA with the streptavidin, the excess SATA was removed again with Zeba^{T M} spin columns and the buffer was changed to PBS with 10 mM EDTA at pH 7.2−7.5. The product solution can be stored frozen at −20^◦C.

For the deacylation a buffer of 0.05 M hydroxylamine in PBS with 25 mM EDTA at pH 7.2−7.5 was done and 100 µl of this buffer was added to 1 ml of the SATA – streptavidin solution. The solution was left to react for two hours in room temperature after which the thiolated streptavidin was ready (22). In the last step the streptavidin has to be cleaned from the remaining unreacted chemicals. This was again done with Zeba^{T M} spin columns.

The success of the thiolation was tested by incubating thiolated strepta-vidin in PBS at pH 7.2 with 10 mM EDTA on freshly evaporated gold

sur-Au CH3

H2N biotin modified DNA

(19)

Figure 4.7: a) On the surface of streptavidin introduced thiol groups are left to react with freshly evaporated gold surface (17) and so the biotin-modified λ -DNA molecules are end-grafted over streptavidin to the surface (18). The thiol modification of the other end of λ - DNA can then be used for double sided end-attachment (19) between two gold surfaces. b) Thiolation of streptavidin:

amino groups on the surface of streptavidin react with SATA yielding the acetyl protected sulphur (20). Deacylation with hydroxylamine (21) activates the thiol group for further reactions (22).

faces and so creating streptavidin coated surface. The unspecific binding sites on the steptavidin coated surface was blocked with BSA. In the final step biotin-modified λ - DNA was incubated with the functionalized surface in TBE at pH 9. With non-thiolated streptavidin the same experiment did not produce any carpet whereas with thiolated streptavidin the results were comparable with streptavidin-glutaraldehyde produced carpets (see Fig. 4.2 (b)).