Tri-Axial Dynamic Acceleration as a Proxy for Animal Energy Expenditure; Should We Be Summing Values or Calculating the Vector?Dynamic body acceleration (DBA) has been used as a proxy for energy expenditure in logger-equipped animals, with researchers summing the acceleration (overall dynamic body acceleration--ODBA) from the three orthogonal axes of devices. The vector of the dynamic body acceleration (VeDBA) may be a better proxy so this study compared ODBA and VeDBA as proxies for rate of oxygen consumption using humans and 6 other species. Twenty-one humans on a treadmill ran at different speeds while equipped with two loggers, one in a straight orientation and the other skewed, while rate of oxygen consumption (VO2) was recorded. Similar data were obtained from animals but using only one (straight) logger. In humans, both ODBA and VeDBA were good proxies for VO2 with all r(2) values exceeding 0.88, although ODBA accounted for slightly but significantly more of the variation in VO2 than did VeDBA (P<0.03). There were no significant differences between ODBA and VeDBA in terms of the change in VO2 estimated by the acceleration data in a simulated situation of the logger being mounted straight but then becoming skewed (P = 0.744). In the animal study, ODBA and VeDBA were again good proxies for VO2 with all r(2) values exceeding 0.70 although, again, ODBA accounted for slightly, but significantly, more of the variation in VO2 than did VeDBA (P<0.03). The simultaneous contraction of muscles, inserted variously for limb stability, may produce muscle oxygen use that at least partially equates with summing components to derive DBA. Thus, a vectorial summation to derive DBA cannot be assumed to be the more 'correct' calculation. However, although within the limitations of our simple study, ODBA appears a marginally better proxy for VO2. In the unusual situation where researchers are unable to guarantee at least reasonably consistent device orientation, they should use VeDBA as a proxy for VO2.
DNase I FootprintingAntonia S. Cardew, Keith R. Fox|Methods in molecular biology|2009 Footprinting is a method for determining the sequence selectivity of DNA-binding compounds in which ligands protect DNA from cleavage at their binding sites. Footprinting templates are typically 50-200 base pairs long, and DNase I is the most commonly used nuclease for these experiments. This chapter describes the preparation and labelling of suitable DNA footprinting substrates, the footprinting experiment itself, and the way in which these data can be used to estimate the dissociation constant of the interaction.
Secondary binding sites for heavily modified triplex forming oligonucleotidesIn order to enhance DNA triple helix stability synthetic oligonucleotides have been developed that bear amino groups on the sugar or base. One of the most effective of these is bis-amino-U (B), which possesses 5-propargylamino and 2'-aminoethoxy modifications. Inclusion of this modified nucleotide not only greatly enhances triplex stability, but also increases the affinity for related sequences. We have used a restriction enzyme protection, selection and amplification assay (REPSA) to isolate sequences that are bound by the heavily modified 9-mer triplex-forming oligonucleotide B(6)CBT. The isolated sequences contain A(n) tracts (n = 6), suggesting that the 5'-end of this TFO was responsible for successful triplex formation. DNase I footprinting with these sequences confirmed triple helix formation at these secondary targets and demonstrated no interaction with similar oligonucleotides containing T or 5-propargylamino-dU.
Stable DNA triple helix formationKeith R. Fox, Antonia S. Cardew, Noemí Vergara et al.|Nucleic Acids Symposium Series|2009 Triplex-forming oligonucleotides bind in the major groove of duplex DNA, generating three stranded structures containing T.AT and C+.GC triplets. Their sequence specific binding has potential uses in gene targeting but is limited by their low affinity, the requirement for low pH and the need for oligopurine targets. We have prepared nucleotide analogues to overcome these limitations and can now target some sequences that contain pyrimidine interruptions at physiological pH. We have tested the biological activity of these modified oligonucleotides. TFOs that contain multiple substitutions with positively charged groups bind with high affinity and we have explored how they interact with secondary sites.
Specificity of triple helix formationAntonia S. Cardew|ePrints Soton (University of Southampton)|2010 Triplex-forming oligonucleotides (TFOs) have been the subject of extensive \nresearch in recent years. They have potential applications in many areas; such as \ngene-based therapies, site-directed mutation and as biochemical tools. However, \ntriplex technology has been hampered by several problems, including low stability \ndue to electrostatic repulsion between strands. This thesis has investigated \ncombinations of four methods for stabilising triplex DNA; these include \nincorporation of the positively charged thymine analogues bis-amino-U and \npropargylamino-dU in TFOs. Also modified TFO’s containing anthraquinone \nderivatives have been tested. Further, the free-intercalating agent \nnaphthylquinoline has been used to modulate TFO binding. \nA TFO containing six consecutive BAU molecules has previously been \nshown to interact with non-target sites. The pH dependence of this TFO was \ninvestigated. These experiments showed that considerably higher TFO \nconcentrations were needed to generate a footprint as the pH was increased. The \nTFO had a high affinity for the exact template (tyrT) at pH 5.0 and 6.0 and showed \nsome evidence of binding even at 30 μM at pH 7.0. These gels also showed \nevidence of the secondary binding seen in previous studies; this was considerably \nmore evident at pH 5.0, however, suggesting that the secondary binding may be \nmore sensitive to pH than the primary binding. \nSecondary binding sites for TFOs were examined by ‘Restriction \nEndonuclease Protection, Selection and Amplification’ or REPSA. REPSA has \nbeen used to select for DNA templates that are bound by the 9mer TFO containing \nsix bis-amino-U residues. Fourteen of the sequences which emerged from \nREPSA were chosen for footprinting with TFOs containing BAU, propargylaminodU \nor T. The BAU-TFO produced clear footprints on all but one of the REPSA \ntemplates tested, indicating that the REPSA process was successful in selecting \nfor sequences which are bound by the TFO. Significantly higher concentrations of \nthe P-TFO were required, and magnesium chloride and / or the triplex binding \nligand naphthylquinoline were needed to promote binding. Despite the differences \nin template sequence there does not appear to be a strong pattern in the binding \nintensities of the TFOs on the different templates. However, all templates do \ncontain a run of four to eight A’s. Surprisingly it appears from these data that the \nBAU TFO discriminates better than the P-TFO against non-exact binding sites \nThe selectivity of TFOs containing anthraquinone modifications was also \ninvestigated. Anthraquinone intercalates between DNA bases in duplex DNA and \ncan be tethered to the end of a TFO to increase stability. The specificity of five \nTFOs with different anthraquinone modifications was examined by footprinting \nagainst fragments containing mismatches. A doubly modified TFO bound with the \nhighest affinity and was most tolerant of mismatches. Mismatches at the centre of \nthe template had a lesser effect on binding affinity than mismatches at the 3’ end. \nThe effect of a 3’ mismatch was also greater if the anthraquinone was at this end. \nThe presence of an S-base at the 3’ end allowing intercalation of the \nanthraquinone at a YpR step increased the binding affinity on the exact template in \ncomparison to TFO 3 which did not contain the S-base. The TFO containing the S \nbase did not bind quite as well as the doubly modified TFO however.