Imaging of RNA in Bacteria with Self-ligating Quenched Probes

Transcription

1 Imaging of RNA in Bacteria with Self-ligating Quenched Probes Shinsuke Sando and Eric T. Kool* Department of Chemistry, Stanford University, Stanford CA CONTENTS: 3 -Phosphorothioate oligonucleotides. (p. 2) Dabsyl- and fluorescein-labeled oligonucleotides. (p. 2) Bacterial strain. (p. 2) Cell fixation. (p. 2) Dabsyl-mediated autoligation reactions on 16S rrna in PFA-fixed E.coli cells. (p. 2) Microscopic imaging and quantitation. (p. 3) Sequences of probe pairs 1-4 and the corresponding target rrnas. (p. 4) E.coli 16S rrna target sequences. (p. 5)

2 3 -Phosphorothioate oligonucleotides. Oligonucleotides containing 3 -phosphorothioate groups were synthesized according to the previously reported method 1, but with the following modifications. Phosphorothioate oligonucleotides were synthesized with DMT-ON method. Following incubation in concentrated ammonia for 12h, DMT groups were deprotected and phosphorothioate oligonucleotides were purified with PolyPakII cartridge (Glen Research). Dabsyl- and fluorescein-labeled oligonucleotides. Dabsyl- and fluorescein-labeled oligonucleotides were prepared as described previously. 1 Briefly, Pac-protected da, ipr-pac-protected dg, and acetyl-protected dc phosphoramidites for ULTRA MILD SYNTHESIS (Glen Research) were employed in synthesizing oligonucleotides containing a dabsyl group. The dabsyl group was added at the 5 terminus by incorporation of 5 -O-dabsylthymidine-3 -O-phosphoramidite. The fluorescein label was introduced with fluorescein-dt phosphoramidite (Glen Research). Deprotection and cleavage from the CPG support was carried out by incubation in 0.05 M potassium carbonate in methanol for 12 h at room temperature. Following incubation, oligonucleotides were purified by reverse-phase HPLC (Allotec BDS-C18 column, 250mm, eluting with 0.1 M triethylammonium acetate ph7.0/acetonitrile). Bacterial strain. For this work, E. coli strain MG1655 was purchased from ATCC. Complete sequences of seven rrs (16S ribosomal RNA) operons (rrs A, B, C, D, E, G, and H) of E. coli MG1655 strain were obtained from the web at GenBank: U Cell fixation. E. coli cells (MG1655 strain) were grown at 37 C in LB broth (DIFCO). When the optical density at 600 nm reached 0.5, the suspension was chilled on ice for 5 min, 1 ml aliquots were taken into 1.5 ml vials, and cells were harvested by centrifugation at 10,000 rpm for 5 min. After centrifugation, supernatant was removed and cells were washed once with 1 ml PBS. To fix the E. coli cells, they were resuspended in 1 ml 4% paraformaldehyde/pbs fixation solution (filter sterilized, ph 8.0 adjusted by 1N NaOH) and the mixture was left at room temperature for 1 h. After fixation, the cells were centrifuged at 10,000 rpm for 5 min, the supernatant was removed, and washed once with 1 ml PBS. After the PBS wash, the fixed cells were resuspended in 1 ml 50% ethanol, and were stored at 20 C. Dabsyl-mediated autoligation reactions on 16S rrna in PFA-fixed E.coli cells. 100 µl aliquots of the fixed E. coli stock suspension were taken into 1.5 ml vials and the fixed cells were collected by centrifugation at 10,000 rpm for 5 min. The cells were washed once with 100 µl PBS, and were resuspended in 100 µl 2

3 hybridization buffer (20 mm Tris-HCl ph 7.2, 0.9 M NaCl, and 0.1% SDS). To the suspension were added 2 µl of 20 µm dabsyl probe and 6 µl of 20 µm phosphorothioate probe. The mixture was incubated at 37 C for 3 h. After incubation, the suspension was directly spotted on glass slide without any washing steps and was covered with micro cover slide. Microscopic imaging and quantitation. Fluorescence images were obtained through an epifluorescence microscope (Nikon Eclipse E800 equipped with 100x objective Plan Fluor apo) with super high pressure mercury lamp (Nikon model HB-10103AF), using a SPOT RT digital camera and SPOT Advanced imaging software. Typical microscope setting is as follows: excitation nm. Typical digital camera settings are as follows: exposure time green 4 sec, binning 2 2, gain 1. For fluorescence quantitation, fluorescence images were converted to gray scale mode using Adobe Photoshop 5.0 software. Grayscale intensities were quantified using NIH Image software. 3

4 Sequences of probe pairs 1-4 and the corresponding target rrnas. target site 1 target site 2 target site 3 target site '-AACGUCGCAAGACCAAAGAGGGGGACC UUCGGGCC-3' 3'-TTGCAGCGTTCTGG TTT CTCCCCC TGGAAGCCCGG- 5' dabsylate 1 S O thioate 1 dab '-AGAGGA UGACCAGCCACAC UGGAACUGAGACACGG UCC-3' 3'-TCTCCTACTGGTCGG TGTGACCTTGACTCTGTGCCAGG -5' S dabsylate 2 O thioate 2 dab '-AACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCA-3' 3'-TTGACTCTGTGCCAGG TCT GAGGATGCCCTCCGTCGT -5' dabsylate 3 O S thioate 3 dab '-AGUCGACCGCC UGGGGAGUACGGCCGCAAGG UUAAAAC-3' 3'-TCAGCTGGCGGACCCC TCAT GCCGGCGTTCCAATTTTG -5' dabsylate 4 S O thioate 4 dab Sequences of probe pairs 1-4 and the corresponding target rrnas. Target rrna, dabsylate probe, and phosphorothioate probe are colored black, green, and red, respectively. T is fluorescein-dt. 4

5 E.coli 16S rrna target sequences (rrs A, B, C, D, E, G, and H). GTCGAACGGTAACAGGAAACAGCTTGCTGTTTCGCTGACGAGTGGCGGACGGGTGAGTAA GTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAA GTCGAACGGTAACAGGAAACAGCTTGCTGTTTCGCTGACGAGTGGCGGACGGGTGAGTAA GTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAA GTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAA GTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAA GTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAA ****************** ******** ** *************************** TGTCTGGGAAGCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCAT ********** ************************************************* AACGTCGCAAGACCAAAGAGGGGGACCCTCGGGCCTCTTGCCATCGGATGTGCCCAGATG AATGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATG AACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATG AACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATG AACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATG AACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATG AACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATG ** ************************ ***************** ************** GGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGA GGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGA GGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA GGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA GGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA GGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA GGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGA ********* ** ******************* *************************** 5

6 6 GGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGGTTGGTTAAGTC ************************************************ *** *******

7 7 AAG-TTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA *** ********************************************************

8 8 TTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGG ****************************************** *** *** ******** ATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTG ************** ***************************************** AAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTTGTTGCCAGCGGTCCGG **************************************** *******************

9 9 *******************************************

10 References 1. Xu Y.; Kool, E. T. Nature Biotechnol. 2001, 19, Sando, S.; Kool, E. T. J. Am. Chem. Soc. 2002, 124,