Determinants of cyclization-decyclization kinetics of short DNA with sticky ends

Cyclization of DNA with sticky ends is commonly used to construct DNA minicircles and to measure DNA bendability. The cyclization probability of short DNA (<150 bp) has a strong length dependence, but how it depends on the rotational positioning of the sticky ends around the helical axis is less clear. To shed light upon the determinants of the cyclization probability of short DNA, we measured cyclization and decyclization rates of ~100-bp DNA with sticky ends over two helical periods using single-molecule Fluorescence Resonance Energy Transfer (FRET). The cyclization rate increases monotonically with length, indicating no excess twisting, while the decyclization rate oscillates with length, higher at half-integer helical turns and lower at integer helical turns. The oscillation profile is kinetically and thermodynamically consistent with a three-state cyclization model in which sticky-ended short DNA first bends into a torsionally-relaxed teardrop, and subsequently transitions to a more stable loop upon terminal base stacking. We also show that the looping probability density (the J factor) extracted from this study is in good agreement with the worm-like chain model near 100 bp. For shorter DNA, we discuss various experimental factors that prevent an accurate measurement of the J factor.

Modeling Deoxyribose Nucleic Acid as an Elastic Rod Inlaid With Fibrils

A classical view of the double-stranded deoxyribose nucleic acid (DNA) as an isotropic elastic rod fails to explain its high flexibility manifested in the formation of sharp loops that are essential in gene regulation and DNA storage. Since the basic structure of DNA can be divided into the external highly polar backbone and the internal hydrophobic bases, here we model DNA as an elastic rod inlaid with fibrils. If the fibrils are much stiffer than the rod, we find that the persistence length of short DNA can be much smaller than that of long DNA with an adapted shear lag analysis. Consequently, the cyclization rate for short DNA is found to be much higher than the previous prediction of the worm-like chain model, which is interestingly in consistency with experiments. Our analysis suggests that the bending of short DNAs can be facilitated if there exists a specific structural heterogeneity.

Steric Effects in the Base-Catalyzed Cyclization of 1-[2-(Methoxycarbonyl)phenyl]-3-(2-substituted Phenyl)triazenes

Eleven model 1-[2-(methoxycarbonyl)phenyl]-3-(2-substituted phenyl)triazenes were synthesized and their cyclization kinetics examined in aqueous-methanolic buffer solutions (51 wt.% methanol) at various pH values. 3(2-Substituted phenyl)benzo[d][1,2,3]triazin-4(3H)-ones were identified as the cyclization products. The log kobs vs pH plot was linear with a slope of unity. Investigation of the steric and electronic effects of substituents in the ortho position revealed that substituents at the ring which is bonded to the N(3) nitrogen affect the cyclization rate through their steric effect only, while their electronic effects are statistically insignificant. This fact was explained in terms of the ring being tilted from the plane in which the remaining part of the conjugate base anion of the model substrate lies. The assumed and confirmed BAc2 mechanism involving specific base catalysis begins by deprotonation of the triazene giving rise to the conjugate base, continues with formation of a tetrahedral intermediate, and ends with elimination of the methanolate ion. Other mechanisms, such as the elimination-addition mechanism via a ketene intermediate or the mechanism involving general base catalysis, are unlikely.

Kinetics and Mechanism of Cyclization of Substituted 2-(Benzoylamino)alkanamides in Strongly Basic Medium

The cyclization reaction of substituted 2-(benzoylamino)alkanamides 1a-1i giving the corresponding substituted 2-phenylimidazol-4(5H)-ones 2a-2i has been studied. The equilibrium constants of reactions of compounds 1d-1f and 2-[(4-nitrobenzoyl)amino]-2,3-dimethylbutanenitrile (3) with methoxide have been determined in methanol-dimethyl sulfoxide media. For compound 1d and N-methyl derivatives 1a-1c, the cyclization rate constants have been measured in dependence on methoxide concentration in media of varying contents of dimethyl sulfoxide. The cyclization reaction mechanism involves formation of anion in a rapid pre-equilibrium and subsequent rate-limiting step: either formation of a cyclic intermediate or splitting off of OH- ion from this intermediate. The product formed in the given medium is immediately transformed into its conjugate base. A change in reaction medium affects the reactions of all the compounds in the same way. The ratio of concentration of substrate to that of its anion at low methoxide concentrations is affected by the solvent composition (MeOH-DMSO). At higher methoxide and DMSO concentrations the reaction rate distinctly decreases, which can be interpreted by the transformation of reactive anion into non-reactive dianion. The corresponding N-methylbenzoylamino compounds are cyclized faster by a factor of 400 as compared with compounds having no methyl group at the benzamide group.

Free-Radical Chemistry of Imines

Aryl radicals bearing an aldimino functional group as part of an ortho substituent cyclized by addition to C and/or N of the imino group. When the choice was between 5-exo closure to C and 6-endo closure to N, the former predominated. However, 6-endo closure to C predominated over 5-exo cyclization to N in isomeric imines. Absolute values of cyclization rate constants were determined and an explanation for the unusual 6-endo preference is offered. Chiral induction in 6-endo cyclization to C of an aldimine from D-glyceraldehyde acetonide was observed, and its sense was determined.

Kinetics of acid-catalyzed cyclization of substituted hydantoinamides to substituted hydantoins

The kinetics of acid-catalyzed cyclization of the hydantoinamides type R3-N(5)H-CO-N(3)R2-CH2-CO-N(1)HR1 (R1, R2, R3 = H and/or CH3) has been studied in 0·5 to 5 mol l-1 hydrochloric acid. The cyclization rate is limited by the rate of the attack of nitrogen atom N(5) on the carbon atom of the protonated amide group. The dissociation constants of the protonated hydantoinamides and rate constants of their cyclizations have been determined. Replacement of hydrogen atom by methyl group at the N(5) nitrogen atom accelerates the cyclization about two times., the same substitution at N(3) accelerates about 50x, whereas at N(1) it results in a 300 fold retardation. With the hydantoinamides having R3 = CH3, the cyclization rate of the protonated hydantoinamide increases with increasing concentration of hydrochloric acid, whereas with the other derivatives this value is independent of the acid concentration.

Mechanism of base-catalyzed cyclization of ethyl N-(substituted aminocarbonyl)glycinates

The cyclization rate constants have been measured of substituted ethyl N-(phenylaminocarbonyl)-, N-(alkylaminocarbonyl)-, and N-(phenylaminothiocarbonyl)glycinates RNHCXNHCH2CO2.C2H5 (X = O, S). Logarithms of these constants increase with decreasing basicity of the amines down to the value of pKa(RNH2) = 5.5. The rate-limiting step of the reaction is formation of the tetrahedral intermediate. With ethyl N-(phenylaminocarbonyl)glycinates (whose pKa(RNH2) values are higher) this dependence, on the contrary, slightly decreases, and the acid-catalyzed splitting off of ethoxy group from the cyclic intermediate becomes rate-limiting. The cyclization rate of a series of ethyl N-(phenylaminothiocarbonyl)glycinates is practically independent of the pKa(RNH2) values, the change in the rate-limiting step would take place at pH about 9.