SMCC Crosslinker: Microtubule Regulation & Bioconjugation

Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC crosslinker) is a non-cleavable and membrane impermeable crosslinker. It contains an amine-reactive N-hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. The maleimide groups of SMCC crosslinker and SMCC are unusually stable up to pH 7.5 because of the cyclohexane bridge in the spacer arm. Because it contains the hydrophilic sulfonyl moiety, Sulfo-SMCC is soluble up to ∼10 mM in water and many commonly used buffers, thus avoiding the use of organic solvents which may perturb protein structure.

SMCC crosslinker Prevents Annealing of Taxol-Stabilized Microtubules In Vitro

Microtubules (MTs) are cytoskeletal filaments that are vital for cell division and vesicle transport in cells. MTs are also believed to play a role in cell mechanics via interconnections with actin filaments. Experiments and simulations have shown that the embedding of microtubules in actin networks lowers Poisson’s ratio of the composite, compared to a pure actin network. MTs are hollow cylinders with a diameter of 25 nm, polymerized from heterodimers of α and β tubulin. Microtubules are polar and have a plus and a minus-end. In the process of constructing such networks, we have here tested the effect of a commercial crosslinker, SMCC crosslinker, on the length distribution of taxol-stabilized microtubules. SMCC crosslinker is a commonly used hetero-bifunctional crosslinker bearing N-hydroxysuccinimide (NHS) ester and maleimide groups to react with primary amines and sulfhydryl groups, respectively. Biopolymer network architecture depends strongly on polymer length, especially for sparse networks. In vitro studies of such networks have been widely used to provide a basis for the understanding of cytoskeletal mechanics. While many experiments have been performed on actin networks, crosslinked with a variety of actin binding proteins (ABPs), networks of crosslinked microtubules have not been much explored. We demonstrate in time-dependent measurements and dual-color experiments that SMCC crosslinker inhibits the end-to-end annealing of stabilized MTs. Curiously, addition of a maleimide or an NHS ester group alone does not show an equivalent inhibition of annealing.[1]

MTs were treated with 250 μM sulfo-SMCC, and imaged after incubation for 0 h, 6 h, and 24 h. Lengths of biopolymers that follow simple polymerization-depolymerization kinetics at fixed rates are distributed exponentially. Consistent with previous studies, both treated and untreated MTs showed exponential length distributions at all time points. The mean lengths of untreated MTs increased significantly after 6 h (p < 0.001) and 24 h (p < 0.001) of incubation, in comparison with those measured at 0 h. We observed that MTs treated with SMCC crosslinker were drastically shorter compared to untreated MTs with almost an order of magnitude difference in their mean lengths. Curiously, maleimide dye treatment at the same concentration resulted in only a two-fold shorter mean length than that of the untreated MTs. It is unclear why the effect of SMCC crosslinker is much more pronounced than that of maleimide dye although they bear the identical reactive group. We therefore tested a control sample to check for a possible effect of the NHS ester group of sulfo-SMCC on MT length. We have shown through systematic experiments that sulfo-SMCC inhibits end-to-end annealing of taxol-stablized MTs. We speculate that the maleimide group on SMCC crosslinker binds to thiol groups on MT ends, and thereby prevents annealing. Previous studies have identified the sulfhydryl (SH) groups of cysteines of tubulin to be essential for MT polymerization.

Synthesis of Peptide-Oligonucleotide Conjugates

Peptide-oligonucleotide conjugates (POCs) possess unique properties that can be exploited in a wide range of applications from nanotechnology to drug delivery and antisense technology (Tung, 2000). Several synthetic protocols have been published that describe different approaches for making POC molecules (Singh, Spinelli et al., 2008). This unit describes the use of SMCC to covalently link amine-modified DNA to cysteine-containing peptides. The protocol was adapted from a previously published method (Harrison and Balasubramanian, 1998), and requires the use of high purity (>95%) amine-modified DNA. A purification method using denaturing polyacrylamide gel electrophoresis (urea-PAGE) is described in Basic Protocol 1. The primary amine modification on the DNA is reacted with the NHS ester moiety of the SMCC crosslinker to attach the bifunctional molecule to the DNA. The SMCC-modified DNA is then conjugated to the polypeptide by a Michael-type addition of the sulfhydryl group of the cysteine residue to the maleimide moiety of the SMCC crosslinker. Peptide-oligonucleotide conjugates can be purified using native-PAGE (Basic Protocol 4) or high performance liquid chromatography (HPLC) (Basic Protocol 5).[2]

The pure amine-modified oligonucleotide is conjugated to SMCC crosslinker through a coupling reaction between the primary amine on the DNA strand and the NHS ester moiety of SMCC. The rate of the reaction is dependent on the pH of the solution (Tournier, Wallach et al., 1998), and must be tightly controlled to avoid cross-reactivity of the amine with the maleimide moiety of the SMCC crosslinker. Optimal buffer conditions are pH 7.0 – 8.0 for both the initial coupling step between the amine-modified oligonucleotide and SMCC reagent, and the second coupling step between the peptide and SMCC-oligonucleotide. As with all synthetic steps, the yield of the final product depends on the purity of the starting materials, with higher purity peptides and DNA giving higher overall yields. Using the methods described above, a yield of 2–6 nmoles of pure POC material can be obtained from native-PAGE purification and 6–10 nmoles from RP-HPLC purification when conducting a 20 nanomole conjugation reaction. The yield may vary depending on the polypeptide sequence and the location of the amine-modified nucleobase in the DNA sequence. To optimize the yield of the reaction, vary the ratio of oligonucleotide to SMCC crosslinker during the first conjugation step and the peptide to SMCC-oligonucleotide during the second conjugation step.

References

[1]Prabhune M, von Roden K, Rehfeldt F, Schmidt CF. Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro. PLoS One. 2016 Aug 25;11(8):e0161623. doi: 10.1371/journal.pone.0161623. PMID: 27561096; PMCID: PMC4999061.

[2]Williams BA, Chaput JC. Synthesis of peptide-oligonucleotide conjugates using a heterobifunctional crosslinker. Curr Protoc Nucleic Acid Chem. 2010 Sep;Chapter 4:Unit4.41. doi: 10.1002/0471142700.nc0441s42. PMID: 20827717; PMCID: PMC2947322.

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