First passage time study of DNA strand displacement

Abstract

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...

Co-transcriptional RNA strand displacement circuits

“…The initial branch migration sequence of the 4_1 gate contains a weak UA tract (Supplementary Fig. 3) that could result in slower strand displacement kinetics (49). This mechanism is consistent with the 4_2r gate reaction being slower than gate reactions with the other three input sequences (Fig.…”

“…20), so the difference in kinetics is not due to the gate misfolding. While all gates reuse the same toehold sequence, the kinetics of the branch migration process can vary over an order of magnitude depending on the sequence (49). The initial branch migration sequence of the 4_1 gate contains a weak UA tract (Supplementary Fig.…”

Kinetics of RNA and RNA:DNA hybrid strand displacement

“…Additionally, toehold location (5 or 3 ) was also predicted to affect the reaction rate due to an extra cross-stacking interaction between the invader and substrate strands when toehold is located at the 5 end, which thus speeds up the reaction. Recently, an experimental study has been carried out where an RNA invades DNA duplex [31]. They compared DNA and RNA single strand invading DNA duplex with long toehold, and found that location of toehold as well as the toehold sequence affect the kinetics of the displacement.…”