Design and synthesis of ASR probe
We derivatized one of the sulforhodamine amino groups with an aromatic moiety aniline, resulting a quenched, non-fluorescent analogue aniline-substituted sulforhodamine (ASR). We hypothesized that the binding of an RNA aptamer would activate the fluorescence emission of ASR (). The second amino group of sulforhodamine was alkylated to introduce a carboxylate group for enabling further biotinylation for immobilization on the beads for in vitro SELEX selection.
Figure 1 Design and synthesis of ASR fluorogenic probes for RNA tagging. a) The general structure of ASR and the scheme for the fluorescence enhancement of ASR by RNA aptamer binding (R1 = CH2CO2−, R2 = Me, R3 = H, R4 = O− for ASR 1 as the SELEX (more ...)
The synthesis of ASR 1
is straightforward as outlined in the (37
). The first step involves the conversion of sulfofluorescein to dichlorosulfofluorscein. Subsequently, one chloro substituent was displaced by aniline, followed by a second substitution by sarcosine to yield the disubstituted sulforhodamine. ASR 1
shows an absorbance maximum at 555 nm and a very low quantum yield in an aqueous buffer (ΦWater
= 0.0017). The mechanism for quenching the sulforhodamine fluorescence emission is believed to operate through photoinduced electron transfer (PET) (39
). The electron in the highest occupied molecular orbital (HOMO) of the aniline group in ASR 1
can transfer to the sulforhodamine HOMO, and prevent the electron at the excited state of sulforhodamine from returning to its ground state via the fluorescence mechanism, resulting in the decrease in the quantum yield of the fluorophore. The PET quenching would become inefficient if the relative geometries of the HOMOs of the fluorophore and aniline are not optimum due to steric restrictions such as the aptamer binding (41
). Consistence with this mechanism, the quantum yield of ASR 1
increases by more than 400-fold to 0.71 in 90% glycerol solution (Supplementary Figure 1
In vitro SELEX and fluorescence screening in E. coli
The selection strategy combines an in vitro SELEX affinity screening () and E. coli fluorescence imaging () to discover aptamers with high binding affinity for ASR 1 and large fluorescence activation.
Figure 2 Discovery of ASR binding aptamers via in vitro SELEX and E. coli fluorescence screening. a) Schematic of the selection strategy. b) Representative fluorescence image of a bacterial plate with 1 μM of ASR 1 in the LB Agar gel. Clones containing (more ...)
A library of 100mer RNA aptamers (estimated to be 1013
) containing 20 constant nucleotides at each end and 60 randomized nucleotide sequences in the middle was transcribed and mixed with the biotinylated analog of ASR 1
) immobilized on the avidin-agarose beads. It was then washed three times with PBS buffer to remove non-bound RNA aptamers, and eluted by excess free ASR 1
, followed by reverse transcription and amplification by PCR. The PCR product was then transcribed for the second round of SELEX. These steps were repeated three times to enrich the RNA sequence with high affinity to ASR 1
Next, the selected pool of RNA aptamers was then screened for their light-up property in E. coli by fluorescence imaging. Out of approximately 200 bacterial plates that were screened, 35 colonies that displayed strong fluorescence signal () were sequenced and contained the following nucleotides at the randomized region: GCAGGACCCT CACCTCGGTG ATGATGGAGG GGCGCAAGGT TAACCGCCTC AGGGTCCTCG
The aptamer containing both this randomized sequence and 20mer primer sequences at each end plus additional flanking sequences for cloning into pBK-CMV-eGFP plasmid vector (HindIII site et al., see Supplementary Table 1 and 2
for the definition and primers) is denoted as Apt10L. The M-fold program predicts 4 secondary structures (), and the 60 nucleotides randomized sequence in Apt10L is predicted to have two possible secondary structures (Supplementary Figure 2a
Figure 3 Sequence and representative secondary structure of selected aptamer Apt10L as predicted by the M-fold program; total 4 structures are predicted for Apt10L. Sequences in blue represent the region of randomized 60 nucleotides. Apt10L contains long flanking (more ...)
The Kd value and the fluorescence enhancement activity of Apt10L for ASR 1 were determined with fluorescence titration (). Fluorescence intensity of ASR 1 at 610 nm increased upon the RNA aptamer binding. The kinetics of binding between the Apt10L and ASR 1 was rather fast: the fluorescence intensities increased instantaneously to the stable values after mixing during the titration. We found that the method used for preparing Apt10L significantly affected the value of Kd. The Kd value of Apt10L for ASR 1 was 39.1 ± 7.6 μM and Fmax (expected fluorescence fold enhancement at saturation) was 135 ± 15 () if Apt10L was purified with G-25 column without denaturing and renaturing subsequent to in vitro transcription. On the other hand, if Apt10L was purified using 6% polyacrylamide-7M urea gel, which is a common method for RNA purification, the value of Kd was 10-fold lower but the value of Fmax was much reduced (Kd = 3.5 ± 1.5 μM, Fmax = 29.0 ± 5.1; ). This result may be explained by the fact that after the PAGE purification Apt10L may not refold into the same conformation formed during in vitro transcription. Considering that the conformation of in vitro transcribed RNA may be closer to its likely structure in vivo, we used G-25-purified RNA apatmers for the binding affinity determination in our study.
Figure 4 Fluorescence titration of Apt10L RNA aptamer against ASR 1 (1 μM) in PBS buffer (pH 7.4) containing 1 mM MgCl2. Excitation and emission wavelength were 555 nm and 610 nm, respectively. a) The Kd value of Apt10L against ASR 1 is 39.1 ± (more ...)
Mutation and truncation analysis to probe the interaction between ASR and Apt10L
To understand the interaction between ASR 1 and Apt10L for further optimization of the binding, a systematic truncation and mutation study was performed. We first designed Apt10M (60mer) with all the flanking sequences but the 60 random sequences removed. In addition, two mutations were introduced at the bottom of its stem region to form two GC base pairs for in vitro transcription and for enhanced stabilization of the stem (). The Kd value of Apt10M for ASR 1 was 75.8 ± 14.3 μM and the estimated Fmax was 125 ± 12.4 (). Compared to Apt10L, a mere two-fold decrease in the binding affinity of Apt10M indicates that the structure of the randomized sequence area is primarily responsible for the binding to ASR 1. On the other hand, the flanking sequences in Apt10L also contribute to both the binding and light-up properties.
Fluorescence titration of Apt10M variants against ASR 1 (1 μM) in PBS buffer (pH 7.4) containing 1 mM MgCl2. Excitation and emission wavelength were 555 nm and 610 nm, respectively.
To examine whether the aptamer binding sequence can be further shortened, Apt10M-54 (54mer) and Apt10M-50 (50mer) variants with three and five less base pairs on the stem regions, respectively, were designed (). The binding affinity of these variants for ASR 1
appears to be dependent on the length of the stem: with a shorter stem, the affinity of Apt10M-50 for ASR 1
decreased by two-fold in comparison to Apt10M, but the affinity of Apt10M-54 for ASR 1
only slightly reduced and the maximal fluorescence enhancement slightly increased (Kd
= 81.2 ± 11.2 μM, Fmax = 136 ± 15.2). On the other hand, further increase in the length of the stem did not necessarily increase the binding affinity and fluorescence enhancement. For example, variants Apt10M-66, -70, -76, -80 (Supplementary Figure 2b
) all showed a lower affinity for ASR 1
than Apt10M except Apt10M-80 that displayed a slightly improved binding affinity but a reduced Fmax value (Supplementary Figure 3a
Both of the two stem-loop structures in Apt10M are required for ASR binding and fluorescence activation. Deleting either stem-loop abolished the binding activity of the mutants (Apt10M-S1 and Apt10M-S2) (). Mutants Apt10M-Lm2 (the size of the right loop was decreased by deleting 5 nucleotides in the middle) and Apt10M-Lm3 (the left loop was replaced by the right loop) displayed undetectable fluorescence enhancement upon the addition of ASR 1, further suggesting the cooperative interaction between the two stem-loop structures ().
Figure 6 Fluorescence titration of Apt10M mutants against ASR 1 (1 μM) in PBS buffer (pH 7.4) containing 1 mM MgCl2. Excitation and emission wavelength were 555 nm and 610 nm, respectively. N.C.: Not Calculable; N.B.: No Binding. Nucleotides in red stand (more ...)
The binding of Apt10M to ASR 1 is highly sensitive to mutations in the stem-loop region. Single or double mutations were introduced to stabilize the representative secondary structures as predicted by the M-fold program, for example, Apt10M1 (containing mutations G27C and A51C), Apt10M2 (containing a mutation C50A), and Apt10M-Lm1 (containing a U39A mutation on the loop region) (). All these mutants either do not bind to ASR 1 or have a largely reduced affinity, suggesting the specific interaction with ASR 1 through the loop.
Influence of the flanking sequence on the binding
The M-fold program predicts several secondary structures for Apt10L (Supplementary Figure 2a
), suggesting that the Apt10L structure is unstable and flexible and that this structural instability could affect its binding affinity to ASR. We examined the role of the flanking sequence on the binding affinity by designing truncated Apt10L variants (T variants). The deletion of some of the flanking sequences decreases the possibility of forming multiple secondary structures. Each of the three Apt10T variants (with the total length of 108, 107 and 88 nucleotides) has similar binding affinity to ASR 1
compared to Apt10L (Supplementary Figure 2c & 3b
), suggesting that the flanking sequences have only an insignificant effect on the binding affinity.
Mutations were also made in the flanking sequence to strengthen the base-pairing on the stems of each conformation of Apt10L predicted by the M-fold program to stabilize these secondary structures. Both Apt10L1 and Apt10L2 (each with 2, or 3 mutations in the flanking sequences, respectively; Supplementary Figure 2a
) showed an identical affinity compared to the Apt10L whereas the Apt10L3 with four mutations showed a 2-fold decrease in the binding affinity (Supplementary Figure 3c
). These results further suggest that the interaction of the flanking sequence to the ASR 1
is not as specific as that in the randomized region, although overall these flanking sequences contribute to a 2-fold increase in the binding affinity.
Improvement of Kd and light-up activity by ASR analogues
To identify the functional groups on ASR 1 that interact with the aptamer, we systematically varied the substitutions on the two amino groups and sulfonate group on ASR 1 to generate a series of analogues. A similar fluorescence titration assay was carried out in the presence of 1 μM of each ASR analogue to estimate its binding affinity with Apt10L ().
Fluorescence titration of Apt10L against ASR analogues (1 μM) in PBS buffer (pH 7.4) containing 1 mM MgCl2. Excitation and emission wavelength were 555 nm and 610 nm, respectively. N.C.: Not calculable; N.B.: No binding.
The elimination of the carboxylate group on ASR 1 and the replacement of the methyl group with a diethyl substitution on the amine led to a 3-fold increase in the binding affinity (Kd = 13.0 ± 1.1 μM, Fmax = 80.6 ± 3.3 for ASR 2). A substitution of the methyl group on the other amine with an ethyl group produces another 2-fold increase in the binding affinity but a largely reduced Fmax (Kd = 6.3 ± 0.5 μM, Fmax = 46.7 ± 1.4 for ASR 3). Interestingly, the introduction of a methoxy group to the quencher moiety, aniline, of ASR 4 abolished the light-up activity, although the quenching effect still remains; it confirms that the quencher moiety provides one of the interaction sites for the Apt10L binding and fluorescence activation.
A long tetra-ethylene glycol group introduced on ASR 5 showed minimal effects on the Kd, which is consistent with the SELEX experiment design that used an ASR analogue biotinylated at the same position as ASR 5. ASR 3 and 6 share similar structures except that the sulfonate group is converted to sulfonamide in ASR 6. This conversion results in a 6-fold decrease in the binding affinity, suggesting that the sulfonate group is also in some contact with Apt10L.
These results suggest that consistent with our design, both methyl aniline group and the sulfonate substituted benzene make important contacts with Apt10L, resulting in the fluorescence activation after binding. Based on these observations, we designed ASR 7 as an improved analogue (), which has a substitution on the amine moiety in the same manner as ASR 2 and a biaryl quencher to increase the contact interface with the aptamer. Indeed, ASR 7 showed a 33-fold increase in the binding affinity for Apt 10L compared to ASR 1 (Kd = 1.2 ± 0.1 μM, Fmax = 88.6 ± 1.7 for ASR 7). In addition, ASR 7 binds tightly to Apt10M with a 2-fold less affinity than Apt10L (Kd = 2.6 ± 0.2 μM, Fmax = 95.1 ± 1.5), as is the case with ASR 1. Despite the highly improved binding affinity by the introduction of a biaryl quencher, ASR 7 does not bind to mutants like Apt10M1 and Apt10M-Lm3, suggesting that the binding specificity was not affected by a biaryl quencher. This result indicates that rational modifications of these functional groups on ASR effectively improve the binding affinity to aptamer and fluorogenic properties.
Figure 8 ASR 7 containing a biaryl quencher has a highly improved binding affinity for Apt10L. Fluorescence titration was carried out in PBS buffer (pH 7.4) containing 1 mM MgCl2. Increasing concentrations of Apt10L RNA or selected Apt10M variants (Apt10M, Apt10M1 (more ...)
Both the binding affinity and fluorescence activation with ASR 7
and Apt10L compare well to previously reported fluorogen-RNA aptamer pairs (31
) whose binding constants range from tens of μM to sub-μM, and whose fluorescence increase is generally less than 100-fold except that malachite green displays 2360-fold. Through mutation and truncation analysis and chemical modifications, we have gained a better understanding of the structural interactions between the ASR fluorogen and the aptamer, which can guide us in continuous optimization to further improve both the binding affinity and the fluorescence activation in this new RNA labeling pair.
In summary, we report here a new small-molecule fluorogen and RNA aptamer pair for RNA labeling. The SELEX procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen. The systematic mutation and truncation study on the aptamer structure determined the minimum binding domain of aptamer specificity to the fluorogen. A series of rationally modified fluorogen analogs have been made to probe the interacting groups of the fluorogen with the aptamer. These results allow us to design ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer compared to original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. The further refining of the probe structure and aptamer selection could eventually lead to an ideal labeling pair that can be used for in vivo RNA live-cell imaging.