Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the [small alpha]-hemolysin ([small alpha]HL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180[degree]. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside [small alpha]-HL are used to derive thermodynamic ([capital Delta]H, [capital Delta]S) and kinetic (Ea) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of [small alpha]HL out of the helix (18 +/- 1 kcal mol-1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the [small alpha]HL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL

The RNA i-motif

International audienc

Laser measurement systems for structural elements’ spacing check

In order to improve monitoring of the provisions of parts during assembly, final operations and subsequent operation the laser measuring system is designed to control the angular position of the structure. In relation to the task, it is necessary to realize a small-sized measuring rotary system providing high accuracy (error of not more than 1.5...2 angular seconds) when significant working distance between the test object and the autocollimator (up to 20 meters) and a relatively wide measurement range (10...12 angular. minutes). The method of "straight angled serifs" was constructed to solve this problem. Measuring channel implements a triangulation method. In accordance with the method of each video camera measures the viewing angles of the laser diode located at the checkpoint. Control surface areas has two phases: the control position of seats held without technological loads and after loading. In the result of the revealed analysis the following primary errors were established to determine the precision measurement of linear and angular coordinates of the test object: the measurement error of the coordinates of the center of the image of the measuring mark on the CCD matrix measuring opto-electronic transducer, due to noise and resolution the receiving area and the measurement uncertainty is determined by the deviation of the focal length of the lens from the nominal value.</jats:p