Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging

Abstract

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson–Crick–Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2′-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme–nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.

Freezing‐Assisted Conjugation of Unmodified Diblock DNA to Hydrogel Nanoparticles and Monoliths for DNA and Hg2+ Sensing

“…To confirm that the DNAwas covalently attached instead of physically trapped, we then analyzed the FAM-A 5 containing hydrogel nanoparticles by gel electrophoresis. [21] This way,o nly the non-covalently associated DNAwould migrate in the electric field. As shown in Figure 2E,lane 1contained the free DNAasacontrol, and lane 2had the gel monomers but without the initiators (prepared by freezing), where the same amount of free DNAw as observed.…”

Chemical Modification and Transformation Strategies of Guide RNAs in CRISPR‐Cas9 Gene Editing Systems

“…Steve D. Knutson et al . developed a glyoxal‐caged gRNA to construct a thermally controlled CRISPR‐Cas9 system [77] . As a thermo‐responsive masking group, glyoxal, reacted with the nitrogen groups on the nucleobases (such as guanosine, adenosine and cytidine) to denature the secondary structure of the guide RNA (Figure 9).…”

“…Steve D. Knutson et al developed a glyoxal-caged gRNA to construct a thermally controlled CRISPR-Cas9 system. [77] As a thermo-responsive masking group, glyoxal, reacted with the nitrogen groups on the nucleobases (such as guanosine, adenosine and cytidine) to denature the secondary structure of the guide RNA (Figure 9). After incubating with glyoxal for 2 h, natural sgRNAs could be effectively protected, thereby completely inhibiting the complex formation of Cas9 protein, sgRNA and target DNA.…”

Recent Advances in Electrochemical Sensors for the Detection of Biomolecules and Whole Cells

“…The detection of small biomolecules (e.g., nucleic acids, enzymes, and hormones) is based on their biological and physiological functions: transmitting genetic information, regulating biological activity, and catalyzing reactions at the cellular level [50][51][52]. However, developing biomolecule sensing technology remains a challenge [53,54].…”