Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green.
In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13-50 nucleotide "guest" RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF "ribosomal RNA aptamer." Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo.
Zhang
Xing
X
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-4004, USA.
Potty
Ajish S R
AS
Jackson
George W
GW
Stepanov
Victor
V
Tang
Andrew
A
Liu
Yamei
Y
Kourentzi
Katerina
K
Strych
Ulrich
U
Fox
George E
GE
Willson
Richard C
RC
D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D002645:Chickens; D049033:Chorioallantoic Membrane; D005453:Fluorescence; D005818:Genetic Engineering; D006801:Humans; D018919:Neovascularization, Physiologic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012394:Rosaniline Dyes / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C005095:malachite green