Overstretching short DNA – Single-molecule force spectroscopy studies

Structural deformation of DNA has a central role in many biological processes. It occurs, for example, during replication, transcription, and regulation of the activity of the genome. To understand these fundamental processes it is necessary to have a detailed knowledge of the mechanical properties of DNA. How DNA responds to longitudinal stress has been studied on the single-molecule level for over two decades. Early it was discovered that torsionally relaxed double-stranded (ds) DNA undergoes a structural transition when subjected to forces of about 60-70 pN. During this overstretching transition the contour length of the DNA increases by up to 70% without complete strand dissociation. Since its discovery, a debate has arisen as to whether the DNA molecule adopts a new form or denatures under the applied tension. In the work of this thesis the overstretching transition is studied using optical tweezers to extend individual dsDNA molecules of 60 – 122 base pairs. By stretching short designed molecules of variable base-composition and with structural modification, factors determining the outcome of the process could be isolated and investigated. The structural changes induced during the transition vary depending on the stability of the dsDNA. Sequences that have a high GC-content are demonstrated to undergo a reversible overstretching transition into a longer form that remains base-paired. At high salt concentrations, this form of DNA, referred to as S-form, is found to be stable for extended periods of time, while at low salt it quickly denatures. AT-rich sequences are found to denature under tension in two different ways: if the AT-rich domain has one free end, melting will occur by progressive peeling of one strand from the other. When peeling is inhibited, here using synthetic inter-strand crosslinks, melting instead occurs internally within the sequence. The results presented here refine our knowledge of DNA mechanics, essential for understanding how proteins in our cells interact with DNA

Force-induced melting of DNA-evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive

Covalent functionalization of carbon nanotube forests grown in situ on a metal-silicon chip

We report on the successful covalent functionalization of carbon nanotube (CNT) forests, in situ grown on a silicon chip with thin metal contact film as the buffer layer between the CNT forests and the substrate. The CNT forests were successfully functionalized with active amine and azide groups, which can be used for further chemical reactions. The morphology of the CNT forests was maintained after the functionalization. We thus provide a promising foundation for a miniaturized biosensor arrays system that can be easily integrated with Complementary Metal-Oxide Semiconductor (CMOS) technology

A stretched conformation of DNA with a biological role?

We have discovered a well-defined extended conformation of double-stranded DNA, which we call Sigma-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change Delta G = 1.57 +/- 0.12 kcal (mol base pair)(-1) 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3'-3' strands, undergo a sharp transition to the 1.52 +/- 0.04 times longer Sigma-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Sigma-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Sigma-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code

Overstretching short DNA – Single-molecule force spectroscopy studies

Structural deformation of DNA has a central role in many biological processes. It occurs, for example, during replication, transcription, and regulation of the activity of the genome. To understand these fundamental processes it is necessary to have a detailed knowledge of the mechanical properties of DNA. How DNA responds to longitudinal stress has been studied on the single-molecule level for over two decades. Early it was discovered that torsionally relaxed double-stranded (ds) DNA undergoes a structural transition when subjected to forces of about 60-70 pN. During this overstretching transition the contour length of the DNA increases by up to 70% without complete strand dissociation. Since its discovery, a debate has arisen as to whether the DNA molecule adopts a new form or denatures under the applied tension. In the work of this thesis the overstretching transition is studied using optical tweezers to extend individual dsDNA molecules of 60 – 122 base pairs. By stretching short designed molecules of variable base-composition and with structural modification, factors determining the outcome of the process could be isolated and investigated. The structural changes induced during the transition vary depending on the stability of the dsDNA. Sequences that have a high GC-content are demonstrated to undergo a reversible overstretching transition into a longer form that remains base-paired. At high salt concentrations, this form of DNA, referred to as S-form, is found to be stable for extended periods of time, while at low salt it quickly denatures. AT-rich sequences are found to denature under tension in two different ways: if the AT-rich domain has one free end, melting will occur by progressive peeling of one strand from the other. When peeling is inhibited, here using synthetic inter-strand crosslinks, melting instead occurs internally within the sequence. The results presented here refine our knowledge of DNA mechanics, essential for understanding how proteins in our cells interact with DNA

Body composition in the elderly: Reference values and bioelectrical impedance spectroscopy to predict total body skeletal muscle mass

BACKGROUND & AIMS: To validate the bioelectrical impedance spectroscopy (BIS) model against dual-energy X-ray absorptiometry (DXA), to develop and compare BIS estimates of skeletal muscle mass (SMM) to other prediction equations, and to report BIS reference values of body composition in a population-based sample of 75-year-old Swedes. METHODS: Body composition was measured by BIS in 574 subjects, and by DXA and BIS in a subset of 98 subjects. Data from the latter group was used to develop BIS prediction equations for total body skeletal muscle mass (TBSMM). RESULTS: Average fat free mass (FFM) measured by DXA and BIS was comparable. FFM(BIS) for women and men was 40.6kg and 55.8kg, respectively. Average fat free mass index (FFMI) and body fat index (BFI) for women were 15.6 and 11.0. Average FFMI and BFI for men were 18.3 and 8.6. Existing bioelectrical impedance analysis equations to predict SMM were not valid in this cohort. A TBSMM prediction equation developed from this sample had an R(pred)(2) of 0.91, indicating that the equation would explain 91% of the variability in future observations. CONCLUSIONS: BIS correctly estimated average FFM in healthy elderly Swedes. For prediction of TBSMM, a population specific equation was required

Bioelectrical impedance spectroscopy in growth hormone-deficient adults.

This study evaluated water compartment assessment by bioelectrical impedance spectroscopy (BIS) by Xitron 4000B in 164 growth hormone-deficient adults on growth hormone replacement therapy, examined the assumed constant body density and gender-specific resistivities in BIS methodology and evaluated a published BMI-adjusted BIS equation. Body composition was measured by BIS, total body potassium (TBK), tritium dilution and dual-energy x-ray absorptiometry (DXA). Tritium dilution and TBK were combined to a reference method for water compartments. Average difference for total body water (TBW) by tritium dilution and by BIS was 0.6 l in women (p > 0.05) and -0.2 l in men (p > 0.05). Average extracellular water (ECW) by the reference method and by BIS differed 1.5 l in women (