In nature, the fusion of phospholipid vesicles is regulated and catalyzed by highly specialized SNARE proteins. A key step in this process is to bring about close apposition of the lipid bilayers that are destined to fuse. Inspired by nature's use of molecular recognition between receptor proteins, we developed a method to force bilayers into proximity by the selective hybridization of membrane-anchored DNA strands. We demonstrate that this forced bilayer contact triggers vesicle fusion.

Intracellular membrane fusion is coordinated by membrane-anchored fusion proteins. The cytosolic domains of these proteins form a specific complex that pulls the membranes into close proximity. Although some results indicate that membrane merger can be accomplished solely on the basis of proximity, others emphasize the importance of bilayer stress exerted by transmembrane peptides. In a reductionist approach, we recently introduced a fusion machinery built from cholesterol-modified DNA zippers to mimic fusion protein function. Aiming to further optimize DNA-mediated fusion, we varied in this work length and number of DNA strands and used either one or two cholesterol groups for membrane anchoring of DNA. The results reveal that the use of two cholesterol anchors is essential to prevent cDNA strands from shuttling to the same membrane, which leads to vesicle release instead of membrane merger. A surface coverage of 6-13 DNA strands was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range. Besides lipid mixing, we also demonstrate DNA-induced content mixing of large unilamellar vesicles composed of the most abundant cellular lipids phosphatidylcholine, phosphatidylethanolamine, cholesterol, and sphingomyelin. Taken together, DNA-mediated fusion emerges as a promising tool for the functionalization of artificial and biological membranes and may help to dissect the functional role of fusion proteins.

A hallmark of DNA polymerases is their inability to synthesize new DNA strands de novoi.e., starting with the polymerization of 2 dNTPs. Rather, DNA polymerases require a stable primer already bound to the template strand in order to synthesize DNA. As discussed below, the primer can be as short as one nucleotide, but more commonly is around 10 nucleotides long. Evolutionarily, this absolute requirement for a primer necessitated the development of cellular machinery to generate these primers. Why can't DNA polymerases initiate new DNA strands de novo?DNA polymerase's requirement for a pre-existing primer appears universal, but why? Potentially, this could result from replication specific issues. For example, a very short primer might increase the number of errors due to difficulties in effectively binding the primertemplate and consequent slippage of the primer in the polymerase active site [1,2]. However, data from RNA synthesis suggest that the requirement is not a replication specific problem. RNA polymerases involved in transcription generate new mRNA strands starting from 2 NTPs. More importantly, RNA replicases, the RNA-dependent RNA polymerases that replicate viral RNAs, also do not require a primer. Since the requirement for a primer extends to replication of viral DNA (Ex., adenovirus and herpes) but not to replication of viral RNA (Ex., influenza and polio), the primer requirement is probably not purely a replication-specific requirement. Alternatively, the requirement could reflect the much higher intracellular concentration of NTPs than of dNTPs [3]. Whereas NTP concentrations vary from around 500 μM for CTP to 4 mM for ATP, dNTP concentrations are only typically 10-100 μM. Since the initiation reaction involves binding of 2 NTPs, using NTPs rather than dNTPs provides a significant catalytic advantage, especially since initiation usually involves 2 purine NTPs whose concentrations are generally higher than those of the pyrimidine NTPs. However, some archaeal primases have the unique ability to synthesize primers using only dNTPs, indicating that the "mass action" effects of higher NTP concentrations cannot be the complete explanation for why primase exists [4,5]. Primer removal and its consequencesAfter completion of DNA replication, the cell must remove the RNA primer and replace it with DNA. This replacement will minimally require a protein to remove the RNA, a DNA polymerase to fill the resulting gap, and a ligase to seal the nick and generate a continuous DNA strand. The transient nature of the RNA primase has two other important consequences -mistakes during primer synthesis will have no effect on chromosomal integrity, and it required the evolution of new proteins for the evolution of linear chromosomes.*To whom correspondence should be addressed. Kuchta@colorado.edu, Phone: 303-492-7027, Fax: 303-492-5894. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscri...

In the present study, we have used the QCM-D technology to study the replication of surface attached oligonucleotide template strands using Escherichia coli DNA polymerase I (Klenow fragment, KF). Changes in resonance frequency (F) and energy dissipation (D) for DNA hybridization and polymerization were recorded at multiple harmonics. Formation of the polymerase/DNA complex led to a significant decrease in energy dissipation, which is consistent with a conformational change induced upon enzyme binding. This interpretation was further strengthened by a data analysis using a Voigt-based viscoelastic model. The analysis revealed a significant increase in shear viscosity and shear modulus during KF binding, whereas the viscoelastic properties of single- and double-stranded templates were almost identical. During the actual DNA synthesis, an initial increase in rigidity (shear viscosity) was followed by a gradual decrease that has two components corresponding to the release of enzyme and to the presence of the catalytically active enzyme/substrate complex. The corresponding decrease in surface concentration was found to underestimate the rate of enzyme release due to viscously coupled water that compensates for the loss in enzyme mass. Furthermore, the modeling elucidates that significant changes in both F and D originate from variations in the viscoelastic properties, which means that changes in F alone should be used with care for estimations of coupled mass and kinetics. Therefore, the modeled temporal variation in effective thickness, being proportional to coupled mass and, thus, independent of structural changes, was used to estimate the catalytic constants of the polymerization reaction. The reported work is the first example providing this type of structural information for the catalytic action of an enzyme, thereby demonstrating the potential of the technique for advanced analysis of complex biological reactions, including proper analysis of enzyme kinetics.