Parallel triplex structure formed between stretched single-stranded DNA and homologous duplex DNA

Chen, Jin; Tang, Qingnan; Guo, Shiwen; Lu, Chao; Le, Shimin; Yan, Jie

doi:10.1093/nar/gkx628

Cited by 15 publications

(11 citation statements)

References 61 publications

(69 reference statements)

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“…Although internal invasion is highly unlikely for the short displacement domain we used here, it would be more probable for longer displacement domains. We also cannot rule out direct swapping between segments of invader and incumbent [63], invasion through triplex formation [64,65], or concurrent dissociation of a weakly bound incumbent strand [32,33]. All these processes can occur in parallel, which makes it difficult to predict the strand displacement rate for any given sequence.…”

Section: Nonuniform Asymmetricmentioning

confidence: 99%

First passage time study of DNA strand displacement

Broadwater

Cook

Kim

2020

Preprint

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6DNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is perva-7 sive in genomic processes and DNA engineering applications. The kinetics of strand displacement 8 have been studied in bulk; however, the kinetics of the underlying strand exchange were obfus-9 cated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence 10 Resonance Energy Transfer (smFRET) approach termed the "fission" assay to obtain the full dis-11 tribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, 12 the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic 13 decay with little delay. Among the eight different sequences we tested, the mean displacement time 14 was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also 15 varied between complementary invaders and between RNA and DNA invaders of the same base 16 sequence except for T→U substitution. However, displacement times were largely insensitive to 17 the monovalent salt concentration in the range of 0.25 M to 1 M. Using a one-dimensional random 18 walk model, we infer that the single-step displacement time is in the range of ∼30 µs to ∼300 µs 19 depending on the base identity. The framework presented here is broadly applicable to the kinetic 20 analysis of multistep processes investigated at the single-molecule level. 21 * These two authors contributed equally. † harold.kim@physics.gatech.edu 1 Nucleic acids' ability to form hydrogen bonds between complementary Watson-Crick 2 bases allows for a rich set of complicated, multi-step kinetic behaviors such as duplex 3 hybridization[1] and dehybridization[2], Holliday junction structural dynamics[3, 4], and 4 strand invasion[5]. In particular, strand displacement, which is the exchange of bases 5 between two competing nucleic acid strands of identical sequence, occurs in homologous 6 recombination[6, 7], DNA replication[8] and RNA transcription[9], as well as CRISPR/Cas[10] 7 and the related Cascade complex[11]. In addition to fundamental genomic processes, DNA 8 nanotechnology exploits strand displacement to create nanoscale gadgets[12-14] and com-9 putational circuits[15-18]. Strand displacement also aids in the development of quantitative 10 assays for detection of nucleic acid[19-21] and enzymatic activity[22, 23] with improved 11 probe specificity[24-26]. 12For practical applications, strand displacement is implemented with the "invader" 13 strand and a partial duplex composed of the "incumbent" strand and the "substrate" 14 strand[18] (Fig. 1). The partial duplex has two distinct domains: 1) the single-stranded 15 overhang called the toehold which is critical to the speed and efficiency of the reaction[27] 16 and 2) the duplex region called the displacement domain. Toehold-mediated strand dis-17 placement is initiated when the invader strand anneals to the toehold in a bimolecular 18 reaction. Once a stable toehold interaction is formed, the incumben...

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Section: Nonuniform Asymmetricmentioning

confidence: 99%

First passage time study of DNA strand displacement

Broadwater

Cook

Kim

2020

Preprint

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“…The ssDNA configuration was excessively applied to study the mechanical properties of ssDNA as well as its interaction with proteins using single-molecule manipulation techniques. − Here, we made it through OSP directly (Figure a). We labeled its 5′ end with a digoxigenin group using the functionalized OSP primer (step i) and labeled its 3′ end with a biotin group using terminal deoxynucleotidyl transferase (TdT) and biotin-dUTP (step ii).…”

Section: Resultsmentioning

confidence: 99%

“…In order to understand the properties and functions at the single-molecule level, various NA configurations have been excessively studied using single-molecule manipulation techniques such as optical tweezers, magnetic tweezers (MT), and atom force microscopy (AFM). , The proteins and other factors have been studied using single-molecule manipulation techniques as well. The manipulated DNA structures include single-stranded DNA (ssDNA), − G-quadruplex, − i-motif, origami, , short hairpin, − double-stranded (ds) DNA with ssDNA handles, replication-fork-shaped (Y-shaped) DNA, − DNA holiday junction (HJ DNA), , three-site multiple-labeled and nicked DNA, − end-opened DNA, − end-closed and torsional-unconstrained DNA, − and torsional-constrained DNA . In these works, repetitive digestion and ligation reactions were usually needed every time the DNA configurations were made, which was very time-consuming and inefficient.…”

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confidence: 99%

A Universal Assay for Making DNA, RNA, and RNA–DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation

et al. 2019

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Despite having a great variety of topologies, most DNA, RNA, and RNA–DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

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“…Such triple-stranded structures (sometimes referred to as “collapsed R-loops”) have been proposed (49,57); for example, it was hypothesized that the displaced non-template DNA strand could be localized in the minor groove of the RNA-DNA hybrid, in which the bases from this strand may form hydrogen bonds with hydroxyl groups from ribose (57). A recent report suggests that mechanically stretched single-stranded DNA can interact with the homologous DNA duplex (58). A similar interaction might occur between the displaced DNA strand and the RNA-DNA duplex within an R-loop, reducing the energetic “cost” for the displaced DNA strand to wind around the RNA-DNA duplex.…”

Section: Mechanistic Aspects Of R-loop Formationmentioning

confidence: 99%

R-loop generation during transcription: Formation, processing and cellular outcomes

et al. 2018

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R-loops are structures consisting of an RNA-DNA duplex and an unpaired DNA strand. They can form during transcription upon nascent RNA "threadback" invasion into the DNA duplex to displace the non-template strand. Although R-loops occur naturally in all kingdoms of life and serve regulatory roles, they are often deleterious and can cause genomic instability. Of particular importance are the disastrous consequences when replication forks or transcription complexes collide with R-loops. The appropriate processing of R-loops is essential to avoid a number of human neurodegenerative and other clinical disorders. We provide a perspective on mechanistic aspects of R-loop formation and their resolution learned from studies in model systems. This should contribute to improved understanding of R-loop biological functions and enable their practical applications. We propose the novel employment of artificially-generated stable R-loops to selectively inactivate tumor cells.

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Parallel triplex structure formed between stretched single-stranded DNA and homologous duplex DNA

Cited by 15 publications

References 61 publications

First passage time study of DNA strand displacement

First passage time study of DNA strand displacement

A Universal Assay for Making DNA, RNA, and RNA–DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation

R-loop generation during transcription: Formation, processing and cellular outcomes

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