Hybridization Kinetics Is Different Inside Cells

Abstract

The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3 0 to 5 0 SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single stranded nucleic acid with bases rU, dU and dT and found that the rate of translocation is dependent upon both base and sugar moieties. We find that the approximate rates of translocation are 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU). These macroscopic translocation rates correspond well to differences in the binding affinity of the translocating NS3h protein to the respective substrates. The values of K M for NS3h translocating at a saturating ATP concentration are: 3.3 (5 0.4) mM nucleotide (poly-dT), 27 (5 2) mM nucleotide (poly-dU), and 36 (5 2) mM nucleotide (poly-rU). Despite the differences in translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation is identical for all three substrates, with a value of ~2 nt per ATP consumed. The identical periodicity of ATP consumption implies a similar mechanism for NS3h translocation along each substrate. This data, together with our independently determined values of K D for NS3h binding to poly-dT (220 5 20 nM nucleotide) and poly-dU (430 5 30 nM nucleotide), suggest that the differences in the macroscopic translocation rates may be explained by differences in the entropic contribution to the binding free energy of NS3h to the different nucleic acid substrates. This conclusion is consistent with observations from a previously published crystal structure of NS3h in complex with a short oligonucleotide (Kim, et al (1998) Structure 6:89-100).

Towards a bioelectronic computer: A theoretical study of a multi-layer biomolecular computing system that can process electronic inputs

“…Alternatively, it may be possible to use methods developed to enhance the speed of biochemical assays involving DNA, such as isotachophoresis 32,33 . Furthermore, it has also been shown that recombination proteins may be able to significantly enhance the rate of hybridization 34,35 , and such proteins may have the potential to speed up DNA computers.…”

MIXING TIME OF A + B− > 0 IN ONE DIMENSION

“…Through Lagrangian dynamics [27], the variation of the energy in the spatial variable x is found by studying the Lagrangian density L = T(x, s) − P(x, s), where T is a kinetic energy and P is a potential energy such that T = (D∂ x c) 2 and P = −sD c 2 .…”

“…If the underlying diffusion is not understood then it is difficult to define the reactivity, whereas the reaction component naturally impedes the diffusivity of the reactants. Consequently, a major goal in experimental and theoretical studies is to elucidate the diffusive or reactive aspects of a scheme which requires understanding, for example; the effect of crowded intracellular conditions on the reduced mobility of a reactant [1,2], the partial differential equation description of a system [3], or the diffusion limit in inhomogeneous environments [4].…”

Real-Time Monitoring of Fluorescence in Situ Hybridization Kinetics

“…13 While hybridization rates obtained in solution or using surface-based methods may provide general guidelines or relative results for a given set of complementary sequences, they cannot assess hybridization rates for large molecules such as chromosomes or an entire genome. 4,14 Further, these methods do not account for factors such as intracellular and intranuclear molecular crowding, 15 spatial arrangement and target accessibility, 16,17 and the significantly slower diffusional transport due to a dense nuclear and chromatin structure, 18,19 and thus cannot provide the appropriate reaction rates of the in situ reaction.…”

“…Other methods suggested for measuring intracellular kinetics include the use of optically switchable probes, such as molecular beacons 27 and temperature oscillation optical lock-in (TOOL) microscopy requiring Förster resonance energy transfer (FRET) probes. 15 However, despite their fundamental importance to FISH assay development, there are currently no techniques for the quantification of the hybridization rates that can be readily applied to any probe or target. Thus the design procedures of FISH probes and assays remain mainly empirical, and the hybridization conditions for a given probe are estimated experimentally using an end-point analysis of the signal.…”