PMID- 16391113
OWN - NLM
STAT- MEDLINE
DCOM- 20060301
LR  - 20231105
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 72
IP  - 1
DP  - 2006 Jan
TI  - Making all parts of the 16S rRNA of Escherichia coli accessible in situ to single 
      DNA oligonucleotides.
PG  - 733-44
AB  - rRNA accessibility is a major sensitivity issue limiting the design of working 
      probes for fluorescence in situ hybridization (FISH). Previous studies 
      empirically highlighted the accessibility of target sites on rRNA maps by 
      grouping probes into six classes according to their brightness levels. In this 
      study, a recently proposed mechanistic model of FISH, based on the thermodynamics 
      of secondary nucleic acid interactions, was used to evaluate the accessibility of 
      the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when 
      thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 
      109 probes were designed with an average thermodynamic affinity (DeltaGo 
      (overall)) of -13.5 kcal/mol. Fluorescence intensity was measured by flow 
      cytometry, and a brightness threshold between classes 3 and 4 was used as the 
      requirement for proof of accessibility. While 46% of the probes were above this 
      threshold with conventional 3-h hybridizations, extending the incubation period 
      to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient 
      thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the 
      low fluorescence intensity of the remaining 14% of the probes. In the end, it was 
      proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a 
      bright probe and, therefore, that there were no truly inaccessible target regions 
      in the 16S rRNA. Based on our findings and mechanistic modeling, a rational 
      design strategy involving DeltaGo(overall), hybridization kinetics, and 
      fluorophore quenching is recommended for the development of bright probes.
FAU - Yilmaz, L Safak
AU  - Yilmaz LS
AD  - Department of Civil and Environmental Engineering, University of 
      Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706-1691, USA.
FAU - Okten, Hatice E
AU  - Okten HE
FAU - Noguera, Daniel R
AU  - Noguera DR
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (DNA, Bacterial)
RN  - 0 (DNA, Ribosomal)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Ribosomal, 16S)
SB  - IM
MH  - DNA, Bacterial/chemistry/genetics
MH  - DNA, Ribosomal/chemistry/genetics
MH  - Escherichia coli/*genetics
MH  - Fluorescent Dyes/chemistry
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Nucleic Acid Conformation
MH  - *Oligonucleotide Probes
MH  - RNA, Ribosomal, 16S/chemistry/*genetics
MH  - Thermodynamics
PMC - PMC1352245
EDAT- 2006/01/05 09:00
MHDA- 2006/03/02 09:00
PMCR- 2006/05/01
CRDT- 2006/01/05 09:00
PHST- 2006/01/05 09:00 [pubmed]
PHST- 2006/03/02 09:00 [medline]
PHST- 2006/01/05 09:00 [entrez]
PHST- 2006/05/01 00:00 [pmc-release]
AID - 72/1/733 [pii]
AID - 1588-05 [pii]
AID - 10.1128/AEM.72.1.733-744.2006 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2006 Jan;72(1):733-44. doi: 
      10.1128/AEM.72.1.733-744.2006.