Real time detection of DNA.RNA hybridization in living cells

Abstract

Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized "molecular beacon" (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in fluorescence intensity in comparison to control MB. By using a fluorescence microscope fitted with UV fluoride lenses, the detection limit of preformed MB/target sequence duplexes microinjected into cells was found to be >/=1 x 10(-1) ag of MB, or approximately 10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB/target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed fluorescence in cells injected with targeted, but not control, MB. These studies suggest that real-time visualization and localization of oligonucleotide/mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.

Figure 1

MB structure. MBs were synthesized with a 24-nt “loop” sequence flanked on the 5′ and 3′ ends with complementary sequence 5 nt long. Internal hybridization of the complementary ends creates the stem–loop structure. The fluorescent donor (EDANS) and acceptor (DABCYL) molecules are joined to the 5′ terminal phosphate and the 3′ terminal hydroxyl group, respectively, through (CH2)6-S-CH2-CO and (CH2)7-NH linker arms.

Figure 2

Specificity of AS MB double helical and hybrid formation in solution. (A–D) Fluorescence emission spectra when various MB sequences were added to solutions of target ODNs. Hybridization was detected by spectrofluorimetry. Excitation wavelength was 336 nm. Signal intensity is displayed along the vertical axis, and the wavelength scanned is displayed on the horizontal axis. An increase in fluorescence intensity, ranging from 15- to 60-fold in comparison to background, was only observed with complementary MB/oligonucleotide pairs. Lowest baselines are tracings derived from solutions containing buffer or ODNs only. (E and F) Spectrofluorimetric tracings derived from AS-MB or SCR-MB, no MB, or MB buffer only incubated with total RNA extracted from K562 hematopoietic cell lines. Addition of MBs complementary to vav (E) or β-actin (F) mRNA sequence resulted in 15- and 9-fold increases in fluorescence intensity respectively in comparison to controls.

Figure 3

Fluorescence emission of MB after microinjection into K562 hematopoietic cells. After injection of 150 μM vav AS or SCR (control) MBs into living K562 human leukemia cells, the cells were examined for signal by phase (A, C, and E) and corresponding fluorescence (B, D, and F) microscopy. Significantly higher levels of cellular fluorescence were observed in cells injected with AS MBs (C and D) than in those cells injected with SCR MBs (A and B). Uninjected cells displayed no fluorescence (E and F). A shows uninjected control cells photographed under phase microscopy; B is the corresponding fluorescent photomicrograph. C and E are AS-MB- and SCR-MB-injected K562 cells, respectively, photographed under phase microscopy, and D and F are their corresponding fluorescent counterparts, respectively. Note that maximal fluorescent emission is found in AS-MB-injected cells.

Figure 4

Relative fluorescence intensity of molecular beacons. K562 cells were microinjected with AS-, SCR-, or NO-β-actin (A) or vav (B) MBs at 150 μM. MBs hybridized to target sequences within 14 min. Cells were then exposed to UV light by using a ×60 plan Apo numerical aperture 1.4 UV fluoride lens. Fluorescent signals were observed within 50 s, and images were captured with a KS-1381 videoscope. Fluorescence levels were measured by using cue series image analysis software with reference to uniformly dyed microspheres.

Figure 5

Confocal image of K562 cells injected with AS vav MBs. Cells injected with AS vav MBs revealed fluorescence signal when irradiated by a laser tuned to excite at 351 nm. Fluorescence images were gathered 15–30 min after MB injection and appeared stronger in the cells’ cytoplasm (outlined by green arrows) than in the nucleus (white arrow), suggesting that hybridization may be favored in the latter location. Uninjected cells or cells injected with control MBs displayed little or no signal and were, therefore, very dark or unseen.