Synthetic m3G-CAP attachment necessitates a minimum trinucleotide constituent to be recognised as a nuclear import signal

Department of Biosciences and Nutrition, Huddinge, Sweden. E-mail: malgorzata.hon Clinical Research Center, Department of La Karolinska University Hospital Huddinge, S Centre of New Technologies, University of Poland Center for Infectious Medicine, Departm Karolinska University Hospital Huddinge, 1 Division of Biophysics, Institute of Experime of Warsaw, Zwirki i Wigury 93, 02-089 War Institute of Bioorganic Chemistry, Polish A 61-704 Poznań, Poland † Electronic supplementary informa 10.1039/c6ra09568b ‡ Authors equally contributed to the work Cite this: RSC Adv., 2016, 6, 51367


Introduction
Accessing the nucleus through the surrounding membrane (nuclear envelope) is one of the major barriers for therapeutic molecules.For medicinal applications, such as plasmid DNA, the large size of the nucleic acid hampers access to the nuclear compartment. 1On the other hand, also for small oligonucleotides (ONs), achieving higher nuclear concentrations by active transport might give the advantage of having a therapeutic effect at lower doses.
The nuclear envelope consists of control gates known as nuclear pore complexes (NPCs), which selectively regulate nuclear transport. 2,3Small molecules such as metabolites and ions can diffuse through the NPC but molecules bigger than 40 kDa such as most proteins and RNA, ribosomal subunits and viral particles need to be actively transported. 4,5The majority of the large molecules have to be either interacting directly with NPCs or be actively transported with soluble carrier molecules collectively named as the karyopherin-b family (importins/ exportins and transportins). 6The Ran-GTP/GDP gradient across the NPC regulates the transport of the cargo molecules between the nucleoplasm and the cytoplasm. 7Ran is a GTPase that can hydrolyse GTP into GDP.RanGTP binds to a carrier importin and helps to release the cargo in the nucleus.Conversely, when a cargo is to be transported to the cytoplasm, exportin-RanGTP-cargo complex is hydrolysed in the cytoplasm releasing the cargo. 8,9arge proteins need to have a nuclear localization signal (NLS) in order to be transported through the NPC.The one most widely studied, is the classical NLS (cNLS) sequence, which contains a short stretch of basic residues. 10,11cNLS containing cargo is recognized by the adapter protein importin-a and the complex is further bound by importin-b that interacts with NPC and translocate the cargo into the nucleus by the help of RanGTP. 12,13On the other hand, the non-classical NLSs do not need an adaptor protein and bind directly to a transporter protein. 6][16][17][18][19][20] Moreover, a more recent study shows the increased transfection efficiency of plasmid DNA by using PEIs (polyethylenimine) conjugated to an NLS peptide in adipose derived stem cell. 21nother NLS type is the 2,2,7-trimethylguanosine cap (m 3 G-CAP) which is naturally found in the uridine rich, small nuclear ribonucleoproteins (U snRNPs) that are involved in pre-mRNA splicing. 22The RNA (snRNA) part of the snRNPs mainly consist of U1, U2, U5 and U4/U6.Among the snRNAs, U1, U2, U4 and U5 are transcribed by RNA polymerase II and co-transcriptionally capped with an m 7 G-CAP.The m 7 G-CAP is recognized by the heterodimeric cap binding complex (CBC) 23 and recruits the phosphorylated adaptor of export protein (PHAX). 24This complex is further recognized by an export receptor CRM1 for cytoplasmic localization and maturation. 24Following the transport, a complex of proteins assemble around the Sm core domain of the snRNAs, 25 which creates a binding platform for trimethylguanosine synthase 1 Tgs1 (PIMT).Tgs1 catalyzes the methylation of the m 7 G-CAP to an m 3 G-CAP. 26,27Subsequently, the m 3 G-CAP is recognized by an adapter protein Snurportin1, which then binds to importin-b for nuclear relocalization, where the nal maturation of the snRNPs occurs. 28,29nvestigation of the crystal structure of Snurportin1 binding domain (residues 97-300) with bound m 3 GpppG, revealed that Snurportin1 binds both the hyper-methylated cap and the rst nucleotide of the RNA in a stacked conformation. 30The investigation of the K d -value for m 3 GpppG dinucleotide (1.0 mM) was determined to be lower than for the m 3 GpppA dinucleotide (12.1 mM), when using the full length Snurportin1.Moreover, binding of full-length Snurportin1 was found to be even stronger (K d ¼ 0.23 mM) for the synthetic capped trinucleotide m 3 GpppA (OMe) U (OMe) A, which more closely resembles the natural UsnRNAs.Since the crystallized domain of Snurportin1 lacked 61 residues from the C-terminus, the authors speculate that the real RNA binding surface of Snurportin1 may be larger and the 2 0 -O-methylated riboses of the RNA stem could have additional interaction. 30reviously, we have reported that use of the m 3 G-CAP as a synthetic RNA 5 0 -end NLS signal increases the nuclear transport of oligonucleotides as well as a cargo protein. 31The use of the m 3 G-CAP as an NLS signal for increased nuclear delivery of a therapeutic compound is likely to require additional stability towards enzymatic degradation.For that reason introduction of stabilizing modications into the triphosphate bridge should be benecial.In addition, the m 3 G-CAP can serve as an adaptor for delivery in vivo, which requires not only convenient synthesis but also a method that is readily scaled up.Thus, we decided to design articially attachable synthetic m 3 G-CAPs, in order to investigate the minimum structural requirements for retained nuclear transport ability.A method for preparation of different constructs was necessary for scanning reaction requirements and allowing attachment of different cargos.We have recently reported the procedure for the synthesis of the m 3 G-CAP analogues equipped with an azide handle that can be conjugated to oligonucleotides via "click reaction". 32Importantly, these m 3 G-CAP-oligonucleotide constructs contain native and modied triphosphate bridges that would stabilize these and reduce degradation. 33Here we report on methodology and synthesis of different m 3 G-CAP-biotin constructs (1-10, Fig. 1), which have been synthesized to evaluate the minimum structural requirement for retained nuclear transport ability in vitro.

Synthesis of m 3 G-CAP-biotin constructs
Different "clickable" m 3 G-CAP constructs (1-8, Fig. 1) with or without triphosphate modications and equipped with an azide handle for attachment to a cargo molecule, were designed and synthesized according to procedures reported earlier. 32,34In addition, a capped trinucleotide construct with a methylene-modied triphosphate bridge and an uncapped trinucleotide control was also synthesized (9 and 10, Fig. 1).
The most simplied structure contains a 2,2,7-trimethylguanosine connected via a triphosphate bridge to the adenosine residue (1).The functional azide linker protrudes from the 2 0 -position of the adenosine nucleoside (structure 1, Fig. 1).Since the 2 0 -O-methyl position may have additional effects on the nuclear transport, m 3 G-CAP construct 2 is equipped with a functional linker at the 3 0 -position.The 2 0 -position is Omethylated, and thus a step closer to the native m 3 G-CAP (structure 2, Fig. 1).The m 3 G-CAP construct 3 is equipped with both a 2 0 -O-methylated adenosine residue and a 3 0 -phosphate as a part of a longer linker attachment.Cap analogues 4-7 contain modication between a and b phosphates of the triphosphate bridge since this can stabilize the m 3 G-CAPs towards degradation. 33,34Finally, the m 3 G-CAP constructs 8 and 9 contain a 2,2,7-trimethylguanosine connected via triphosphate bridge to the AUA sequence, where A and U are 2 0 -O-methylated (structure 8 and 9, Fig. 1).In 9 the same methylene modication of the triphosphate as in 4 is included.The 5 0phosphorylated trimer (structure 10, Fig. 1) can also be attached to cargos and used as a non-capped control of conjugates in the studies of nuclear transport.
Synthesis of 9 and 10 was performed as reported in the synthesis of 8 with the exception that for 10 the capping step was performed with the methylene(bisphosphonate) analog of N 2 ,N 2 ,N 7 -trimethylguanosinepyrophosphoryl imidazolide.In This journal is © The Royal Society of Chemistry 2016 order to assess the nuclear transport with a sizeable cargo, the different cap constructs were attached via linkers to biotin that in turn can be bound to a uorescent streptavidin cargo (STV-Alexa488).From our experience in conjugating ONs to peptides it seems that low concentration of the reacting biomolecules requires more active functional groups to achieve sufficient conversion. 35Thus, biotin constructs with different linkers attached to an activated triple bond donor, p-(N-propynoylamino)toluic acid PATS, 35 were synthesized (11, 12 36 and 13, Fig. 2).Synthesis of m 3 G-CAP-biotin and uncapped trinucleotide constructs (Scheme 1) were performed in solution using a tertbutanol/water mixture as solvent using Cu(I) catalyzed Hüisgen dipolar [3 + 2]-cycloaddition ("click chemistry") (Scheme 1) which resulted in the products 14-26 (Table 1).

Biological evaluation of synthetic m 3 G-CAP-biotin constructs
The synthesized m 3 G-CAP-biotin analogues were then evaluated with respect to localization within cells.Streptavidin-Alexa488 (STV-488) was incubated with 3.5 to 4 molar excess of the constructs 14-26.STV has a molecular weight of ca.56 kDa.Together with the biotinylated constructs the total molecular weight adds up to 60-70 kDa, which is well above the threshold for passive diffusion through the nuclear pore channels (NPCs).For evaluation of possible nuclear localization, STV-complexes with 14-26 were transfected into U2OS cells by the PULSin protein transfection reagent.Aer 6 hours, cells were analyzed by uorescent microscopy in order to detect the localization of the STV-Alexa488 complexes.
All constructs with the m 3 G-CAP connected to a mononucleotide, whether the triphosphate was modied or not, failed to show up in the nucleus (see ESI †).However, cells treated with the construct having a capped trinucleotide (23)  and to a lesser extent the same cap construct conjugated to biotin with a different linker (26) displayed uorescence signals from the nucleus in the form of speckles (Fig. 3), in line with our previous publication where longer oligonucleotides were equipped with biotin linkers at the 3 0 -end. 31omewhat surprising is that no nuclear localization was observed (Fig. 3) with the m 3 G-CAP carrying a methylene(bisphosphonate)-modied triphosphate bridge even when this was connected to a trinucleotide (24).As expected control cap analogues 22, 25 and STV-Alexa488 alone showed no nuclear accumulation (Fig. 3).
As a complementary study we also ran confocal microscopy in order to conrm that the origin of the signal comes from the nucleus.For this study we decided to compare the compound 23 to control 22.Using 3D image processing soware, we dened the volume of the nucleus based on the nuclear staining.Fig. 4 shows the confocal microscope image to the le, and the processed image with the dened nuclear volume and the signal coming from STV-Alexa488 within that volume to the right.Cells treated with STV-Alexa488 complex with 23 showed a clear signal within the dened nuclear volume, whereas control 22 did not.

Conclusions
That, none of the "shorter" mononucleotide m 3 G-CAP-biotin constructs seem to end up in the nucleus (caps 1-7, constructs 14-21) despite the fact that these "short" CAPs bind well to Snurportin1 37 suggests that more is required for nuclear transport than just Snurportin binding.It is not clear why the methylene(bisphosphonate) modied m 3 G-capped trinucleotide is not transported, but suggests that it there is a sensitivity even to minor alterations.These studies demonstrate that Fig. 3 Fluorescence microscopy images of the cells treated with STV-Alexa488 complexes with constructs 22-26 (Table 1).Nuclear region of the cells are marked with a white arrow.Nuclear transport is evaluated by the Alexa-488 signal from the nucleus.efficient transport of a cargo is highly sensitive to the exact design of the Snurportin1-interacting m 3 G-CAP.Owing to that the only structure, which shows the interaction 30 is based on a truncated form of Snurportin1, it is not straightforward to predict the conformations, which are tolerated, let alone to use this structure as a basis for further improvements.Thus, while the existing structure can be used as a starting point, currently the only way to study this interaction is by testing new constructs in biological assays.It should also be mentioned that simply enhancing the binding interaction may not directly predict biological activity, since apart from binding to Snur-portin1, interaction with importin b and the release of the cargo may be equally important.One could speculate that this could be related to the interaction of the snurportin1-complex with importin b when this is recruited to obtain nuclear transport. 38n any case, our studies clearly demonstrate that not only the m 3 G-CAP, but also the adjacent nucleotides are crucial for efficient nuclear transport within cells.The ndings suggest that a minimum requirement is three nucleotides connected to the CAP and nuclear transport could well be more efficient with even longer oligonucleotide attached to the m 3 G-CAP.Further studies on such constructs would be valuable for further exploration of m 3 G-CAPs as adaptors for transport of therapeutic cargos into the nucleus.

Confocal microscopy
For the confocal analysis, glass microscope cover slips were coated with poly-lysine solution (Sigma Aldrich, 0.1% (w/v)) for 1 hour at RT. Subsequently, 50 000 cells were plated onto coated cover slips in a 24-well plate.6 hours post-transfection with STV-CAP complexes, the cells were xed using 4% PFA for 10 min at 37 C. Then cells were washed with PBS 1Â twice.Aer this, DRAQ5 nuclear stain (Abcam) was added onto the slides and incubated for 5 min at 37 C. Aer washing the cells with PBS 1Â twice, the cells were mounted using ProLong Antifade mounting medium (ThermoFisher Scientic).Images were obtained on a Nikon A1R confocal system with a 60Â/1.49oil objective, acquired using NIS-Elements A1R soware (Version 3.2; Nikon Instruments) and analyzed with Imaris 3D image processing soware (Bitplane).

Fig. 2
Fig. 2 Structures of biotin linkers used in the study.

Fig. 4
Fig. 4 Confocal microcopy analysis of the cells treated with complexes of STV-Alexa488-and m 3 G-CAP biotin conjugates 23 or with control 22. Left panel shows the confocal image, whereas the right panel shows the defined nuclear volume and the STV-Alexa488 signal coming from within that volume.