Protocol for sortase-mediated construction of DNA–protein hybrids and functional nanostructures

Abstract

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes.

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

“…16 When physical adsorption is unavailable or undesirable, more involved covalent conjugation chemistries are needed for high force application. 13,[17][18][19][20][21][22][23] Furthermore, the high force regime is outside the practical range of many standard approachesfor example, optical tweezers can run into problems with available laser power, local heating, or free radical generation, and magnetic tweezers typically do not have strong enough field gradients to generate these forces with standard magnetic microspheres. 1,24,25 These problems limit investigations in the high force regime under conditions of constant force or loading rates slow enough to explore nearequilibrium behaviour.…”

How to Perform miRacles: A Step‐by‐Step microRNA Detection Protocol Using DNA Nanoswitches

“…To make the nanoswitch, the first step is to linearize the circular M13 scaffold strand, followed by hybridization with backbone oligonucleotides and detector strands. Depending on the purpose of detection, the nanoswitches can be optionally purified by PEG precipitation (Support Protocol 1; Koussa, Sotomayor, & Wong, 2014)…”

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

“…Notable examples include the ligation of proteins or peptides to fluorophores (Popp et al, 2007; Yamamoto & Nagamune, 2009), carbohydrates (Samantaray, Marathe, Dasgupta, Nandicoori, & Roy, 2008; Wu, Guo, Wang, Swarts, & Guo, 2010), polymers (Parthasarathy et al, 2007; Qi, Amiram, Gao, McCafferty, & Chilkoti, 2013), solid supports (Chan et al, 2007; Le, Raeeszadeh-Sarmazdeh, Boder, & Frymier, 2015; Sinisi et al, 2012), therapeutics (Beerli, Hell, Merkel, & Grawunder, 2015; Fang et al, 2016), lipids (Antos et al, 2008; Wu et al, 2010), nucleic acids (Koussa, Sotomayor, & Wong, 2014; Pritz et al, 2007), metal chelators (Paterson et al, 2014; Westerlund, Honarvar, Tolmachev, & Eriksson Karlström, 2015), viral particles (Hess et al, 2013; Schoonen et al, 2015), live cells (Popp et al, 2007; Shi et al, 2014; Yamamoto & Nagamune, 2009), or even intramolecular ligations for generating cyclic proteins and peptides (Antos et al, 2009b; Jia et al, 2014; van ’t Hof et al, 2015). A full discussion of all relevant applications is beyond the scope of this unit, and we refer to the reader to reviews on this topic (Haridas, Sadanandan, & Dheepthi, 2014; Popp & Ploegh, 2011; Ritzefeld, 2014; Schmohl & Schwarzer, 2014).…”