The effect of nucleotide substitution on DNA denaturation profiles.

The melting profiles were obtained for DNA restriction fragments of approx. 1150 bp with deletion of one, five or six base pairs making them different from each other. In all cases the deletions caused a shift of one melting peak without affecting the positions of the other three peaks. The effect amounted to 0.28 +/- 0.03C upon the deletion of one GC pair. The melting of DNA fragments was also studied by electrophoresis in denaturing gradient gels. The deletion of one GC pair was shown to cause an appreciable shift of the electrophoretic denaturation profile.     

Early melting of supercoiled DNA.

Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.     

Localization of low-melting regions in phage T7 DNA

Specific fragmentation of T7 DNA at glyoxal-fixed denatured regions by the S1 endonuclease followed by restriction analysis made it possible to localize four low-melting regions in phage T7 DNA. These regions have the following coordinates:0.5-1.2;14.8+/-0.3;46.3+/-0.5; 98.4+/-0.3 (in T7 DNA length units). The location of the low-melting regions was refined by means of electron-microscopic denaturation mapping and gel electrophoresis of partially denatured DNA. The obtained localization of the low-melting regions is consistent with the available data on the sequence of T7 DNA. The map of low-melting regions was compared with the genetic map of T7 DNA.Images     

The nonequilibrium character of DNA melting: effects of the heating rate on the fine structure of melting curves.

To investigate the effects of heating rate on the DNA melting profile and to test the predictions of the theory of slow relaxation processes in DNA melting (1) concerning these effects, we obtained differential melting curves for the Bsp I C1 fragment of T7 DNA (1461 bp) and the Sma I-Eco RI fragment of Col E1 DNA (1291 bp) at heating rates of 0.05 and 0.5 deg/min. At low ionic strength (0.02 M Na+) the heating rate has been shown to affect the position of the third peak in melting curve for C1 fragment. According to the melting maps (2), this peak corresponds to the unwinding of the section between the end of the molecule and the region already melted. At high ionic strength (0.2 M Na+), when the melting of this DNA is reversible (3), the position of the peaks does not depend on the heating rate. In the case of the Col E1 DNA fragment the heating rate affects, as might be expected from the melting maps (4), only the last peak, as the melting of the last section is always nonequilibrium. The results of the study are in good qualitative agreement and in satisfactory quantitative agreement with the theoretical predictions (1).     

Specific fragmentation of T7 phage DNA at low-melting sites.

A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of 13.6 +/- 0.4, 8.2 +/- 0.4 and 3.5 +/- 0.16 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determined by means of analysis of the intermediates during SI-cleavage.     

A comparison of experimental and theoretical melting maps for replicative form of phi X174 DNA

A previously elaborated technique for fixing a chosen partially melted state of DNA with glyoxal was used in a study of the melting process of the replicative form (RF III) of phi X174 DNA. Electron-microscopic maps corresponding to five points of the melting curve of RF III were obtained and compared with the theoretical melting maps obtained in (4) and (6). This comparison clearly shows that only rigorous calculations (4) and not the ones proposed by Azbel (6,7) correctly predict the course of RF III melting.     

Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.     

The Kinetics of DNA Helix-Coil Subtransitions

Abstract

The kinetic analysis of individual helix-coil subtransitions was performed by comparing melting and renaturation profiles obtained at different temperature change rates. The duration of the three transition stages and its dependence on temperature and ionic strength were determined for a T7 phage DNA fragment. The obtained temperature dependence of the melting time for a stretch flanked by melted regions is in quantitative agreement with that predicted by the theory of slow processes (V.V. Anshelevich, A.V. Vologodskii, A.V. Lukashin, M.D. Frank-Kamenetskii, Biopolymers 23, 39 (1984)). The reasons are discussed for the increasing relaxation time of this stretch in the middle of its transition with decreasing ionic strength.

The zipping kinetics of a melted region under essentially nonequilibrium conditions was examined for T7 fragment and pAO3 DNAs. The obtained temperature dependence of the zipping time is in quantitative agreement with calculations based on the theory of slow processes.

The renaturation times of stretches flanked by helical regions proved fairly small even at a low ionic strength. These times are several orders of magnitude smaller than the renaturation times of the same stretches with one helical boundary. A formal application of the theory of slow processes failed to account for the small renaturation times of stretches that are zipped from both ends. This is probably due to the non-allowance for the changing entropy of the loop linking two helix-coil boundaries migrating towards each other.

Slow processes have been revealed in the intramolecular melting of Col E1 DNA at a high ionic strength. The reason for the long relaxation time of one subtransition is the large size of the loop that separates the melting stretch from the helical part of the molecule. This result can be accounted for by the theory of slow processes.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.