ON-OFF switching of transcriptional activity of large DNA through a conformational transition in cooperation with phospholipid membrane

Journal of the American Chemical Society

Volumn 132, Issue 35, 2010, Pages 12464-12471

  Tsuji, Akihiko ^a   Yoshikawa, Kenichi ^a,b  

^a JAPAN SCIENCE AND TECHNOLOGY AGENCY   (Japan)

^b KYOTO UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

AQUEOUS PHASE; AQUEOUS SOLUTIONS; ARTIFICIAL CELL; CONDENSING AGENTS; CONFORMATIONAL CHANGE; CONFORMATIONAL TRANSITIONS; DISCRETE CONFORMATIONAL TRANSITION; DNA MOLECULES; FOLDED CONFORMATION; FOLDED STATE; INNER SURFACES; MEMBRANE SURFACE; MICRO DROPLETS; MRNA MOLECULES; OLIGONUCLEOTIDE PROBES; PHOSPHATIDYLETHANOLAMINE; PHOSPHOLIPID MEMBRANE; POLYAMINES; STRUCTURAL TRANSITIONS; TRANSCRIPTIONAL ACTIVITY; WATER-IN-OIL;

DNA; GENES; MEMBRANES; MOLECULES; MONOLAYERS; OLIGONUCLEOTIDES; PHOSPHOLIPIDS; SOLUTIONS; TRANSCRIPTION;

CONFORMATIONS;

AMINE; DOUBLE STRANDED DNA; MAGNESIUM; MESSENGER RNA; SPERMINE;

ADSORPTION; AQUEOUS SOLUTION; ARTICLE; ARTIFICIAL MEMBRANE; CELL FUNCTION; CONCENTRATION (PARAMETERS); CONFORMATIONAL TRANSITION; DNA TRANSCRIPTION; FLUORESCENCE MICROSCOPY; OLIGONUCLEOTIDE PROBE; PHOSPHOLIPID MEMBRANE; SURFACE PROPERTY; TRANSCRIPTION INITIATION;

DNA; HUMANS; NUCLEIC ACID CONFORMATION; PHOSPHOLIPIDS; SURFACE PROPERTIES;

EID: 77956252296     PISSN: 00027863     EISSN: 15205126     Source Type: Journal    
DOI: 10.1021/ja105154k     Document Type: Article

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45. For λZAPII/actin DNA, ∼30000 transcription sites in a 40 μm microdroplet correspond to 43 ng/μL of DNA. It is actually difficult to encapsulate such high concentration of long λZAPII/actin DNA into microdroplets.
46. As described above, the folding transition of long DNA is a first-order phase transition that is discrete at individual DNA molecule level. (7, 8) Therefore, in the intermediate spermine concentration region, DNA molecules with a coiled conformation and those with a folded conformation coexist. The results and interpretation in Figure 5 are consistent with this fact.
47. The average numbers of discrete FRET signals were not zero, but rather 0.27 and 0.20 at spermine concentrations of 0.6 and 0.75 mM, respectively. At these spermine concentrations, we observed several microdroplets that had only one signal. It is possible that the DNA was cut off to a shorter length during the experimental handling. If the cut-off DNA was too short to exhibit a conformational transition to a folded state, transcription might occur on this DNA (see later discussion on 2.1 kbp DNA). Therefore, we interpret our results to indicate that "none of the (long λZAPII/actin) DNA was "active" at more than 0.6 mM of spermine".
48. We encapsulated the same concentration (0.25 ng/μL) of 2.1 kbp DNA in microroplets as was used for λZAPII/actin DNA; 0.25 ng/μL of 2.1 kbp DNA corresponds to more than 30000 molecules in a microdroplet with a diameter of 40 μm. Indeed, there were a large number of discrete FRET signals in the microdroplets. In addition, the diffusion of discrete signals in the aqueous phase was faster than in the case of λZAPII/actin DNA. Therefore, it was actually difficult to count the number of discrete FRET signals in the microdroplets.
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