Rational design of hybrid DNA–RNA triplex structures as modulators of transcriptional activity in vitro

et al. 2023

Advanced Materials

The compaction and organization of genomic DNA is a central mechanism in eukaryotic cells, but engineered architectural control over double‐stranded DNA (dsDNA) is notably challenging. Here, long dsDNA templates are folded into designed shapes via triplex‐mediated self‐assembly. Triplex‐forming oligonucleotides (TFOs) bind purines in dsDNA via normal or reverse Hoogsteen interactions. In the triplex origami methodology, these non‐canonical interactions are programmed to compact dsDNA (linear or plasmid) into well‐defined objects, which demonstrate a variety of structural features: hollow and raster‐filled, single‐ and multi‐layered, with custom curvatures and geometries, and featuring lattice‐free, square‐, or honeycomb‐pleated internal arrangements. Surprisingly, the length of integrated and free‐standing dsDNA loops can be modulated with near‐perfect efficiency; from hundreds down to only six bp (2 nm). The inherent rigidity of dsDNA promotes structural robustness and non‐periodic structures of almost 25.000 nt are therefore formed with fewer unique starting materials, compared to other DNA‐based self‐assembly methods. Densely triplexed structures also resist degradation by DNase I. Triplex‐mediated dsDNA folding is methodologically straightforward and orthogonal to Watson‐Crick‐based methods. Moreover, it enables unprecedented spatial control over dsDNA templates.

Section: Discussionmentioning

confidence: 99%

Folding Double‐Stranded DNA into Designed Shapes with Triplex‐Forming Oligonucleotides

et al. 2023

Advanced Materials

“…Interestingly, many DNA-modifying proteins target the major groove of dsDNA, but this accessibility is blocked in densely triplexed structures, thereby altering native enzymatic activity towards these structures. Transcription can also be regulated by triplex formation and recently it was demonstrated how engineered transcription units are regulated by hybrid RNA-DNA triplex structures formed at the promoter 57 .…”

Section: Discussionmentioning

confidence: 99%

Folding double-stranded DNA into designed shapes with triplex-forming oligonucleotides

et al. 2023

Preprint

The folding of double-stranded DNA around histones is a central mechanism in eukaryotic cells for compacting the genetic information into chromosomes. Very few artificial methods are available for controlling the shape of dsDNA at any level, whereas several artificial methods have been developed to efficiently organize single-stranded DNA and RNA into a variety of well-defined nanostructures by programmed self-assembly. Here, we show how long double-stranded DNA sequences can be spatially organized by triplex-forming oligonucleotides (TFOs), which bridge two or more encoded polypurine domains. The linearized or plasmid dsDNA is compacted into antiparallel folds, which enables the formation of raster-filled 2D shapes and 3D structures with either square or hexagonal organizations. Contrary to ssDNA, dsDNA has inherent rigidity which alleviates the requirement to saturate a structure with TFO strands, yet the TFOs are still able to bend the dsDNA controllably and steeply up to 180° over 6 bp. The majority of structures investigated here are formed by Hoogsteen interactions which require pH = 5-6, however, the methodology is also applied with reverse Hoogsteen interactions at physiological pH. In both cases, the DNA triplexes render pure polypurine scaffolded structures resistant to DNase I.

Section: Discussionmentioning

confidence: 99%

Folding double stranded DNA into designed shapes with triplex forming oligonucleotides

et al. 2022

Preprint

The folding of double-stranded DNA around histones is a central mechanism in eukaryotic cells for compacting the genetic information into chromosomes. Very few artificial methods are available for controlling the shape of dsDNA at any level, whereas several artificial methods have been developed to efficiently organize single-stranded DNA and RNA into a variety of well-defined nanostructures by programmed self-assembly , . Here, we show how long double-stranded DNA sequences can be spatially organized by triplex forming oligonucleotides (TFOs), which bridge two or more embedded polypurine domains. The linearized or plasmid dsDNA is compacted into antiparallel folds, which enables the formation of raster-filled 2D shapes and 3D structures with either square or hexagonal organizations. Contrary to ssDNA, dsDNA has inherent rigidity which alleviates the requirement to saturate a structure with TFO strands, yet the TFOs are still able to bend the dsDNA controllably and steeply up to 180° over 6 bp. The majority of structures investigated here are formed by Hoogsteen interactions which require pH = 5-6, however, the methodology is also applied with reverse Hoogsteen interactions at physiological pH. In both cases, the DNA triplexes render pure polypurine scaffolded structures resistant to DNase I.