Next-generation PNA chimeras exhibit high affinity and potent gene

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Next-generation PNA chimeras exhibit high affinity and potent gene silencing Alexandre J Debacker, Vivek K. Sharma, Pranathi Meda Krishnamurthy, DANIEL O'REILLY, Rachel Greenhill, and Jonathan Watts Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00827 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 8, 2018

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Biochemistry

Next-generation PNA chimeras exhibit high affinity and potent gene silencing Alexandre J. Debacker1,2, Vivek K. Sharma1, Pranathi Meda Krishnamurthy1, Daniel O’Reilly2,3, Rachel Greenhill2, Jonathan K. Watts1,4* 1RNA

Therapeutics Institute, UMass Medical School, Worcester, MA 01605, USA; 2Department

of Chemistry, University of Southampton, Southampton SO17 1BJ, UK; 3Current address: Department of Chemistry, McGill University, Montreal, QC, H3K 2K7, Canada; 4Department of Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, MA, 01605, USA. *Email: [email protected]; Phone: +1 (774) 455-3784; Fax: +1 (508) 856-6696

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract We present a new design of mixed-backbone antisense oligonucleotides (ASOs) containing both DNA and peptide nucleic acid (PNA). Previous generations of PNA-DNA chimeras showed low binding affinity, reducing their potential as therapeutics. The addition of a 5'-wing of locked nucleic acid as well as the combination of modified nucleotide and PNA monomer at the junction between PNA and DNA gave high affinity to the chimeras. The resulting ASOs demonstrated high serum stability and elicited robust RNase H-mediated cleavage of complementary RNA. These properties allowed the chimeric ASOs to demonstrate high gene silencing efficacy and potency in cells, comparable with LNA gapmer ASOs, both by lipid transfection and gymnosis.


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Biochemistry

Introduction Antisense oligonucleotides (ASOs) are emerging as a major class of therapeutics. Four FDAapproved ASOs are on the market and dozens more are in clinical trials. This has largely been made possible by the evolution of ASO chemistry.1-3 A key enabling modification has been the phosphorothioate (PS) backbone, which increases both nuclease stability and cellular uptake.4 Nevertheless, the PS backbone is also associated with toxicity.5 “Mixed-backbone” oligonucleotides containing a mixture of PS and non-PS linkages may provide a path to exploit the favorable uptake properties of PS modified ASOs while minimizing their toxicity. As such, several groups have recently published examples of ASOs containing PS and PO (i.e. unmodified) linkages.6-9 An even better approach might be to combine PS linkages with other modified linkages to maintain high nuclease stability in combination with reduced toxicity. The neutral oligonucleotide analogue peptide nucleic acid (PNA)10 shows high affinity to complementary DNA and RNA10-12, excellent resistance to enzymatic degradation13 and improved ability to bind structured targets relative to other modified oligonucleotides.14 The original PNA design is based on an aminoethylglycine backbone (aeg-PNA).10 A number of groups have also developed modified PNA backbones, including chiral monomers and various sidechains.15-16 Nevertheless, PNA is unable to recruit RNase H, shows relatively low solubility17 and has poor membrane permeability,18-19 limiting its bioavailability. An approach to address both the limitations of PNA and of fully PS oligonucleotides would be to make chimeric oligomers combining PNA and PS-DNA. Four groups independently described DNA-PNA chimeras in the late 1990s.20-24 These chimeras were able to recruit RNase H to cleave complementary RNA in vitro.25-26 Incorporating PNA at the 3'-end of the oligomer also increased 3'-exonuclease resistance up to 50-fold relative to unmodified DNA.20 However, these chimeras showed relatively low affinity for their targets: while PNA normally increases binding affinity by 1-2°C per base, the melting temperatures reported for DNA-PNA chimeras were similar to those obtained for DNA/DNA duplexes.23-29 We hypothesize that this low affinity of DNA-PNA chimeras for RNA comes from tension created by the two parts of the chimeras that adopt different helical structures or backbone trajectories. Supporting this hypothesis, a mismatch at the junction between DNA and PNA is less destabilizing than in any other position.20, 22 Nevertheless, this is likely to be a small structural effect since PNA/RNA duplexes have a helical structure similar to that of DNA/RNA duplexes.27, 30-31 Thus, the key to improving the RNA-binding affinity of PNA chimeras may be to optimize the design of the junction, and a relatively subtle change may be effective.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In this work, we have addressed the low affinity of previous generations of PNA chimeras by including a series of conformationally distinct nucleotides at the junction of the PNA and DNA portions of the oligomer. We show that the optimal length of the first PNA monomer depends on the nature of the linking nucleotide, as was previously observed for a different series of PNAcontaining chimeras.32-33 Using this optimized junction, we then describe a new gapmer design in which one wing is PNA and the other is LNA, characterize their biophysical properties, and demonstrate that they have potent gene silencing ability in mammalian cells. Experimental Methods Oligonucleotide synthesis, purification and characterization The PNA part of the chimera was synthesized at 2-µmol scale using an Expedite 8909 synthesizer, on Tentagel-OH® solid support (Novabiochem) functionalized with Fmoc-glycine or MMT-aminohex-1-yl. MMT-AdeBz, MMT-Thy, MMT-CytBz were purchased as a custom product from Link Technologies. MMT-GuaiBu and all three DMT-Thy PNA linkers were synthesized in house as described in the supporting information. All monomers were dissolved to 0.2M in anhydrous N-methylpyrrolidinone. Coupling time was 8.5 min using HATU (Alfa Aesar) as activator and a DIPEA/lutidine mix as base; double coupling was performed on selected bases and 3% TCA in DCM (Sigma Aldrich) was used as deblock solution. The oligonucleotide part was synthesized using standard oligonucleotide methods at 1-µmole scale on an Applied Biosystems 394 DNA synthesizer. BTT (0.25 M in acetonitrile, ChemGenes) was used as activator. 0.02 M iodine in THF/water/pyridine (ChemGenes) was used as oxidizer. Sulfurization was accomplished with DDTT (0.1 M, ChemGenes) using a 1 min wait time. 3% TCA in DCM (TEDIA) was used as deblock solution. LNA phosphoramidites were synthesized in-house from the 3'-hydroxy nucleoside precursor (Rasayan Inc.) using standard methods,34-35 and were dissolved in acetonitrile (or for LNA-CBz, DCM) to 0.15 M. DNA and RNA phosphoramidites (ChemGenes) were dissolved in acetonitrile to 0.1 M and 0.15M, respectively. Coupling time was 10 min for RNA and LNA and was 40 seconds for DNA. In most cases, oligonucleotides were grown on 1000 Å CPG functionalized with Unylinker (~42 µmol/g). Chimeras and oligonucleotides were deprotected with concentrated aqueous NH4OH (1 mL) at 55 °C for 16 h. Note that chimeras are not stable to methylaminecontaining solutions. Then the oligonucleotides were evaporated to dryness in a centrifugal evaporator and resuspended in 1 mL RNase-free water. For RNA-containing oligonucleotides, the TBDMS deprotection was achieved with DMSO/NEt3•3HF (4:1) solution (500 µL) at 65 °C for 3 h. RNA oligonucleotides were then recovered by precipitation in 3M NaOAc (25 µL) and n-BuOH


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Biochemistry

(1 mL), and the pellet was washed with cold 70% EtOH and resuspended in 1 mL RNase-free water. Purification of oligonucleotides were carried out by high performance liquid chromatography using a 1260 infinity system with an Agilent PL-SAX 1000 Å column (150 x 7.5 mm, 8 µm). Buffer A: 30% acetonitrile in water; Buffer B: 30% acetonitrile in 1M NaClO4 aq. Excess salt was removed with Sephadex Nap-25 column. Chimeras used in UV-melting and CD experiments were purified a second time using 20% polyacrylamide gel electrophoresis. Oligonucleotides were analyzed on an Agilent 6530 Q-TOF LC/MS system with electrospray ionization and time of flight ion separation in negative ionization mode. The data were analyzed using Agilent Mass Hunter software. Buffer A: 100mM hexafluoroisopropanol with 9 mM triethylamine in water; Buffer B: 100mM hexafluoroisopropanol with 9 mM trimethylamine in methanol. RNase H assays RNase H assays were conducted in 200-µL PCR tubes with E. coli RNase H (NEB, 5000u/mL). The solutions of FAM-labeled RNA (5µM in water) and ASO (5µM in water) were prepared beforehand. A solution of 1/10 Enzyme in 1x reaction Buffer (75 mM KCl, 50 mM Tris-HCl, 3 mM MgCl2, 10 mM dithiothreitol) was prepared and kept on ice. For 1:5 ASO:RNA studies: on ice, 65 µL of water and 8.4 µL of 10x reaction buffer were added to a 200-µL PCR tube. Then solutions of ASO (1.6 µL, 5µM) and prepared 1/10 enzyme solution (8 µL) were added. The reaction was started by addition of of RNA solution (8 µL, 5 µM) is the start of the assay. The zero aliquot (10 µL) was removed immediately, mixed with formamide (10 µL), and kept at -20°C. The reaction was maintained at RT protected from light, and subsequent 10 µL aliquots were treated identically. After the reaction, the samples at -20°C were taken and rapidly heated to 75°C for 5 min to ensure total enzyme inactivation. For each time point, 10 µL of the reaction-formamide mix was mixed with 10 µL of RNA Sample Loading Buffer (Sigma Aldrich), then 15 µL of the sample was loaded on a denaturing 20% polyacrylamide gel and run at 400V. The gel was read with a GE -Typhoon FLA 9500 gel imager on the FAM channel. The relative intensity of the gel bands was quantitated with ImageJ and normalized to the intensity of the band at t=0. FBS assay To 20 µL of oligonucleotides at 5 µM in 45 µL of water and 10 µL of 10xPBS, 25 µL of fetal bovine serum was added. The mixture was incubated at 37°C and 10 µL was taken out at 0, 5min, 15min, 30min, 1h, 6h and 24h, mixed with 10 µL of formamide and kept at -20°C. Then 1 µL of proteinase K mix was added to the time point sample and the sample is incubated at 55°C for 10 min. Then 10 µL sample were loaded on 15% polyacrylamide TBE urea gel. The gel was


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

visualized by staining with sybr gold and read with a GE -Typhoon FLA 9500 gel imager on the sybr gold channel. Cell culture and transfection The mouse embryonic fibroblast (MEF) WT cells and mouse hepatoma cells (Hepa 1-6) were purchased from ATCC and cultured in DMEM supplemented with 10 % FBS. Cells were maintained in a 5% CO2-humidified incubator. For lipid transfection, MEF cells were plated in 96 well plates at 8 K cells/well at least 16 h prior to transfection. Cells were transfected with nontargeting controls, PNA chimeras and LNA gapmers targeting Malat1 using Lipofectamine RNAiMAX (Life Technologies). Cells were harvested 16 h post transfection and Malat1 and Hprt RNA levels were measured using Quantigene 2.0 assay (Affymetrix,#QS0011 Mouse Hprt: SB15463 and Mouse Malat1: SB-26581) following the manufacturer’s protocol. For gymnotic delivery of chimeras and LNA gapmers, Hepa 1-6 cells or MEFs were plated at 11k cells/well in 12 well tissue-culture-treated plates. Cells were treated with oligonucleotides at 5 different concentrations in complete media. Cells were harvested after 5 days and assayed for Malat1 and Hprt RNA levels using the Quantigene 2.0 assay. Results and Discussion Chimera Design To address the low affinity of previous PNA chimeras, we set out to optimize the DNA-PNA junction. We focused on both the 3'-nucleotide of the DNA and the first PNA monomer (called the PNA linker since it contains a linking oxygen instead of a nitrogen). We included either DNA, LNA or 2'F-ANA at the junction (Figure 1c-e). DNA is relatively flexible, while 2'F-ANA is less flexible with a preferred south-east conformation,36 and LNA is rigidly north (RNA-like). These nucleotides are each coupled with three different types of PNA linker that differ in the length of their backbone (based on hydroxyethyl, hydroxypropyl or hydroxybutyl glycine, Figure 1b). We hypothesized that varying the length of the PNA linker might yield appropriate combinations in combination with nucleotides of different conformation and flexibility: thus considering the final nucleotide and first PNA monomer as a unit, this allowed us to test nine different junctions.


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Biochemistry

a)

b)

B O

O N

H2N

O

OH

HO

O

B

B

O c)

Thy O

O

O N OH n n = 1, 2, 3

B

O

O d)

O

O O

e)

F

O

Figure 1: Chemical structures of a) PNA monomer; b) PNA linker; c) DNA; d) LNA ; e) 2'F-ANA. B = nucleobase and Thy is thymine.

Figure 2: Design of chimeras used in this study. Letters A-E indicate which sequences (Table 1) are based on each design architecture. Sequences A-D were used for biophysical and enzymatic studies, while sequence E was a Malat1-targeted oligomer used for gene silencing studies in cells.

We synthesized chimeras of three principal designs (Figure 2, Table 1). To optimize the linkage affinity, we first synthesized a series of 15mer oligomers consisting of 8 DNA, one nucleotide linker of variable chemistry, one PNA linker of variable length, and five aeg-PNA monomers (Figure 2a); we applied this design to compare junction affinity across three sequences. We then synthesized a series of 17mer oligomers which further contained a 5'-end “wing” of three locked nucleotides (Figure 2b) and used these to study both binding affinity and RNase H activity in vitro. These first designs made use of normal phosphate linkages in the LNA and DNA portions of the oligomer. Finally, we synthesized a series of 19mer oligomers containing PS linkages in the LNA and DNA portions of the oligomer and used them to study silencing activity in cells. Synthesis In order to synthesize the chimeras, we adapted an in-line solid phase strategy using N-MMTprotected PNA monomers with base-labile nucleobase protecting groups.20, 23 Accordingly, MMT/Bz PNA-A, MMT/Bz PNA-C, and MMT-PNA-T monomers were purchased from Link


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Technologies. MMT/iBu PNA-G monomer and PNA linkers were synthesized in house (see Supporting information). The PNA end of the chimera was synthesized on functionalized tentagel on an Expedite 8909 synthesizer, using HATU as activator. The PNA-containing solid phase was then transferred to an ABI 394 DNA synthesizer to grow the nucleotide part of the oligonucleotide using phosphoramidite chemistry. Deprotection was performed with concentrated aqueous ammonia at room temperature for 48 hours and chimeras were then purified by anion exchange chromatography. Expected Mass

Observed Mass

(g/mol)

(g/mol)

PO

4484.4

4484.2

ACG TAT GG+A et acatc

PO

4512.4

4512.2

ACG TAT GGA pt acatc

PO

4498.4

4497.3

A_Ch_pLNA

ACG TAT GG+A pt acatc

PO

4526.4

4526.2

A_Ch_bDNA

ACG TAT GGA bt acatc

PO

4512.4

4512.4

A_Ch_bLNA

ACG TAT GG+A bt acatc

PO

4540.4

4540.2

B_Ch_eDNA

ACG TAT GGC et tcaac

PO

4460.3

4459.2

B_Ch_eLNA

ACG TAT GG+C et tcaac

PO

4502.4

4501.2

C_Ch_eDNA

ACG TAT GGT et ttttt

PO

4488.4

4892.7

C_Ch_eLNA

ACG TAT GG+T et ttttt

PO

4515.4

4514.2

C_Ch_pDNA

ACG TAT GGT pt ttttt

PO

4501.4

4500.1

C_Ch_pLNA

ACG TAT GG+T pt ttttt

PO

4529.4

4529.1

C_Ch_bDNA

ACG TAT GGT bt ttttt

PO

4515.4

4515.2

C_Ch_bLNA

ACG TAT GG+T bt ttttt

PO

4543.3

4543.3

D_Gaps_LNA

+G+T+A CGT ATG G +T+T+A

PS

4365.5

4364.4

D_Ch_eDNA

+G+T+A CGT ATG GT et acatc

PO

5236.9

5236.5

D_Ch_pDNA

+G+T+A CGT ATG GT pt acatc

PO

5251.0

5250.4

D_Ch_bDNA

+G+T+A CGT ATG GT bt acatc

PO

5265.0

5264.4

D_Ch_eLNA

+G+T+A CGT ATG G +T et acatc

PO

5265.0

5264.4

D_Ch_pLNA

+G+T+A CGT ATG G +T pt acatc

PO

5279.0

5278.5

D_Ch_bLNA

+G+T+A CGT ATG G +T bt acatc

PO

5293.0

5292.5

D_Ch_eFANA

+G+T+A CGT ATG G fT et acatc

PO

5254.6

5254.5

D_Ch_bFANA

+G+T+A CGT ATG G fT bt acatc

PO

5283.0

5282.5

E_MALAT1_Ch_LNA

+G+G+T CAG CTG CCA +A et gctag

PS

6059.1

6059.0

E_MALAT1_Ch_DNA

+G+G+T CAG CTG CCA A et gctag

PS

6031.1

6031.0

E_MALAT1_Gap_LNA

+G+G+T CAG CTG CCA +A+T+G

PS

4985.9

4985.5

Name

Sequence (5′ to 3′ / N to C)

Backbone

A_Ch_eDNA

ACG TAT GGA et acatc

A_Ch_eLNA A_Ch_pDNA

Table 1: List of the chimeras and gapmers synthesized for this study. “N” is DNA, “+N” is LNA, “n” is PNA, “et” is ethyl linker, “pt” is propyl linker and “bt” is butyl linker. The first letter of the name (A-E) reflects the base sequence. Additional control oligonucleotides are shown in Supporting Table S1.


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Biochemistry

Binding Affinity The affinity of the different chimeras was measured by UV melt on duplexes with fully complementary RNA oligonucleotides. The first set of melting experiments (Table 2) were conducted on 16-mer chimeras consisting of 8 DNA nucleotides at the 5'-end, one additional nucleotide (either DNA or LNA) at the junction, then a PNA linker and 5 normal PNA monomers at the 3'-end (Figure 2). Three different sequences were included in this study to confirm that the effect of the junction on affinity was independent of the oligonucleotide sequence. To facilitate interpretation of our work we have given these compounds intuitive names: A, B, and C refer to the base sequence. PO_DNA and PO_RNA represent fully DNA and RNA sequences, respectively, used as controls. Ch indicates that the oligomer is a chimera; e, p and b represent the ethyl, propyl and butyl PNA linker at the junction while the DNA, LNA or 2'F-ANA in these sequences refers to the chemistry of the nucleotide at the junction. Sequence A

Tm (°C)

ΔG310

Sequence B

Tm (°C)

(kJ/mol)

ΔG310

Sequence C

Tm (°C)

(kJ/mol)

ΔG310 (kJ/mol)

A_PO_DNA

41.1 ± 0.1

-42.7 ± 0.5

B_PO_DNA

46.6 ± 0.5

-49.3 ± 1.7

C_PO_DNA

38.5 ± 0.2

-39.4 ± 0.3

A_PO_RNA

55.1 ± 0.3

-52.2 ± 0.9

B_PO_RNA

57.3 ± 0.0

-55.5 ± 0.8

C_PO_RNA

44.6 ± 0.2

-46.4 ± 1.0

A_Ch_eDNA

39.5 ± 0.9

-39.2 ± 0.2

B_Ch_eDNA

45.4 ± 0.3

-42.6 ± 0.5

C_Ch_eDNA

42.4 ± 0.9

-40.3 ± 0.6

A_Ch_pDNA

43.4 ± 0.1

-46.5 ± 0.7

C_Ch_pDNA

47.3 ± 0.2

-42.3 ± 0.1

A_Ch_bDNA

43.3 ± 0.7

-45.9 ± 1.9

C_Ch_bDNA

46.9 ± 0.7

-41.6 ± 0.4

A_Ch_eLNA

44.6 ± 0.6

-45.3 ± 1.0

C_Ch_eLNA

50.2 ± 0.3

-43.9 ± 0.4

A_Ch_pLNA

44.2 ± 0.3

-45.2 ± 0.4

C_Ch_pLNA

49.8 ± 0.2

-43.0 ± 0.1

A_Ch_bLNA

41.6 ± 0.6

-43.5 ± 0.4

C_Ch_bLNA

47.4 ± 0.3

-42.2 ± 0.3

B_Ch_eLNA

50.3 ± 1.0

-46.6 ± 0.3

Table 2: Melting temperature of the different chimera designs against complementary RNA. A, B and C represent different sequences (details in Table 1). Duplexes are at 1 µM in 1xTRIS buffer with 140 mM NaCl. Tm and ΔG310 are reported as mean ± standard deviation of n=3. Samples were heated at 1 °C/min and temperatures were calculated with the hyperchromicity method from Agilent Thermal software.

Table 2 shows that the modifications have the same impact on the melting temperature, regardless of the sequence. The design that has been most widely studied in the past (Ch_eDNA) shows the lowest melting temperature. The substitution of the DNA by LNA at the junction greatly improves the Tm (more than +5°C) when ethyl linker is used. However, the substitution of DNA by LNA when using propyl and butyl linker has little to no effect (+/- 2°C) The use of propyl linker (+3-5°C) and butyl linker (+4°C) in combination with DNA improves the affinity. These results underline the importance of compatibility between the PNA linker and the nucleotide at the junction: they have to be approached as one entity, since the optimal linker in the context of one nucleotide can be destabilizing in the context of a nucleotide with a different conformation.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Melting studies were also performed on the chimeric gapmer (Figure 2, Design D) which includes a wing of LNA at the 5'-end (Table 3). The variations observed in the Tm of the chimeric gapmers are similar to the ones observed for the previous design with similar junctions. The substitution of DNA by LNA in combination with an ethyl linker (compare eDNA to eLNA) greatly increased the Tm (+3°C). The use of propyl and butyl linkers with DNA (pDNA and bDNA) improved the Tm (+2°C) while it slightly lowered the Tm (–1-2°C) if it was used with LNA (pLNA and bLNA compared to eLNA). When the linking nucleotide is 2'F-ANA, the ethyl linker shows a Tm equivalent to that of eDNA. However, coupled with butyl linker, bFANA shows a high Tm (55.7°C), nearly as high as eLNA. This underlines again that the junction has to be considered as a whole when investigating the best chemistry for stability. It is interesting that the combination used in most previous reported studies (eDNA) shows the lowest Tm. Our results are consistent with work by Greiner et al. in which optimal linker length was different for a DNA-PNA chimera than for a [2'OMe-RNA]-PNA chimera.32-33

In summary, after testing nine combinations, the eLNA and bFANA chimeras

showed the highest binding affinity. Design

Tm (°C)

ΔG310 (kJ/mol)

D_PO_DNA

49.3 ± 0.8

-46.5 ± 1.2

D_PO_RNA

63.9 ± 0.1

-59.6 ± 1.3

D_PS_DNA

48.0 ± 0.5

-47.2 ± 0.7

D_Gap_LNA

62.9 ± 0.1

-59.6 ± 2.9

D_Ch_eDNA

52.4 ± 0.6

-56.0 ± 0.7

D_Ch_pDNA

54.7 ± 0.2

-55.6 ± 0.7

D_Ch_bDNA

52.7 ± 0.2

-47.4 ± 0.7

D_Ch_eLNA

56.5 ± 0.4

-53.4 ± 1.2

D_Ch_pLNA

55.2 ± 0.1

-53.8 ± 0.4

D_Ch_bLNA

53.0 ± 0.3

-51.4 ± 0.8

D_Ch_eFANA

53.1 ± 0.2

-55.6 ± 1.3

D_Ch_bFANA

55.7 ± 0.1

-56.1 ± 1.2

Table 3: Melting temperatures of chimera:RNA duplexes at 1µM in 1xPBS. Steps are 0.5˚C/min against RNA complementary strand. Tm, and ΔG310 were obtained using the hyperchromicity method within the Agilent Thermal software. Mean and standard deviation were calculated from n=3. Full sequences are given in Table 1.

Circular Dichroism To further understand the properties of the chimeric gapmers, we investigated the helical structure of the duplexes formed by chimeras and RNA by circular dichroism (CD). The chimeras used in these studies are the D series of sequences, for which melting data is shown in Table 3.


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100

RNA/RNA

eFANA eLNA eDNA

DNA/RNA

DNA/DNA

50

Molar ellipticity (mdeg.cm-2.µmol-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0 250

300

350

wavelength (nm)

-50

Figure 3: Circular dichroism spectra of ASO/RNA duplexes. CD performed at 1 µM duplex in 1xPBS at 4°C with 5 mm path, using the D series of sequences (see Tables 1, 3 and Figure 2).

All the chimera:RNA duplexes adopt an A-type helical structure, with CD spectra intermediate between an A-form RNA:RNA helix and an A-like RNA:DNA helix as seen in Figure 3. The A-form RNA:RNA duplex shows a characteristic positive peak at 260nm and negative peak at 210nm. The DNA:RNA hybrid duplex shows a positive peak of slightly lower intensity at 265nm and a negative peak at 210nm, as expected for a modified “A-like” helical structure.. All chimeras show a positive peak at 260nm which is lower in magnitude than the A-form RNA homoduplex but of similar magnitude to the DNA:RNA hybrid. Thus the PNA chimera appears to adopt an A-form or A-like helix with complementary RNA. Additional CD spectra are shown in Supporting Figures S1–S4. RNase H Activity The different chimera designs were tested for their ability to elicit RNase H cleavage of a complementary FAM-labelled RNA target (Figure 4, Supporting Figures S5–S8).

RNase H

requires a footprint of at least 7 base pairs of DNA-RNA hybrid duplex, but prefers 8 in the context of a PNA chimera,37 and the gap in our chimeras is 7 or 8 nucleotides. We first tested a singlestranded, unstructured RNA target using multi-turnover conditions (5 equivalents of target RNA,


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4). Overall, the different chimera designs show similar activity in this RNase H assay. The assay does not show a correlation with binding affinity or identify one chimera design as better than the others. Similar results were obtained using single-turnover conditions (i.e., a ratio of 1:1 ASO:RNA, Figure S5). We wondered whether the PNA tail would provide an advantage when binding structured targets, since the uncharged nature of PNA has been previously observed to improve binding/invasion of structured target. Therefore, we constructed a FAM-labeled RNA target that contained a hairpin structure (Supporting Figures S7-S8). The PNA chimeras were able to elicit RNase H-mediated cleavage of this structured target, but their neutral tail did not appear to give them a kinetic advantage over an isosequential LNA gapmer ASO, even for this structured RNA target.


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a

b 100

50

_G ap s D _C _LN h A D _eD _C h NA D _pD _C h_ NA b D _C DN h_ A D _C eLN A h D _pL _C N D h_ A _C b h LN D _eF A _C A h_ NA bF A N A

0

D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4: a) RNase H assay with 5 equivalents single stranded FAM-labeled complementary RNA target. b) Quantitation of full-length RNA remaining after this RNase H assay. Time points are (control, 0 , 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h), mean of 2. The full gels are shown in Supporting Figure S6.

Serum Stability To assess the stability of our chimeras to serum nucleases, we incubated the oligomers with 25% FBS and monitored disappearance of the full length oligonucleotide over time. Comparison of the


Biochemistry

stability of the chimera eDNA to PO DNA is striking (Figure 5). Under these conditions the phosphodiester DNA is degraded in less than 15 minutes while for the chimera a significant amount of full length oligonucleotide is still present after 24 h, a striking result in light of the fact that this chimera still contains a phosphodiester backbone. This result confirms the high nuclease resistance of our LNA-DNA-PNA gapmer design, and is consistent with 3'-exonuclease activity

24 h

6h

1h

30 min

5 min 15 min

0

being the predominant serum nuclease activity.38

Ref

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

D_PO_DNA

D_Ch_eDNA Figure 5: Stability of PO-DNA and PO-containing chimeric ASOs to serum nucleases. ASOs were incubated at 37˚C in 25% FBS (20% PAGE, visualized by SYBR Gold).

Gene silencing in mammalian cells To be successful gene silencing agents inside cells, ASOs need to compete with target selfstructure and protein binding. Thus, high affinity correlates with high potency for ASOs,39-40 at least up to a threshold.41 Since the eLNA chimera design showed good RNase H activity and high binding affinity, we selected this junction for study of gene silencing activity in cells. We included the classic (eDNA) chimera for comparison; to the best of our knowledge, no prior group has published data on the gene silencing acitivity of PNA chimeras in cells. For both junction designs, we synthesized these as LNA-DNA-PNA chimeras (i.e. gapmer ASOs with one wing being LNA and the other PNA). Our ASOs contained a DNA gap of 9 nucleotides and a phosphorothioate backbone throughout the LNA and DNA portions of the oligomer (Figure 2, Design E). The gapmers were designed to target Malat1 RNA. We first transfected the chimeric ASOs into cells using a cationic lipid (Lipofectamine RNAiMAX). We compared our chimeric oligomers to LNA gapmers, which represent a gold standard for ASO


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potency, and included non-targeting controls of both designs. Both types of chimeras showed potency and efficacy comparable to the LNA gapmers, with 50% silencing observed at ~1 nM (Figure 6). MEF 200 150

NTC_Gap_LNA NTC_Ch_DNA

100

E_MALAT1_Gap_LNA

50

E_MALAT1_Ch_DNA E_MALAT1_Ch_LNA 0 0. 8 1. 6 3. 1 6 12.3 .5 25 50

0 0. 8 1. 6 3. 1 6 12.3 .5 25 50

0 0. 8 1. 6 3. 1 6 12.3 .5 25 50

0 0. 8 1. 6 3. 1 6. 12 3 .5 25 50

0 0 0. 8 1. 6 3. 1 6 12.3 .5 25 50

Malat1 RNA (% of control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[ASO] (nM)

Figure 6. PNA-DNA-LNA chimeras silence Malat1 expression after lipid transfection into mouse embryonic fibroblasts. RNA levels were quantified using the QuantiGene 2.0 assay, and normalized to Hprt.

The presence of a neutral PNA tail might affect cellular uptake or subcellular trafficking. Naked or “gymnotic” delivery shows a better correlation with in vivo performance than lipid transfection.42 Therefore, we tested the activity of our PNA chimeras by gymnotic delivery in two cell lines (Figure 7). In gymnotic delivery, the ASO is added into the growth media of the cells and taken up into the cells by endocytosis. In this experiment, MEFs and Hepa 1-6 cells were treated with ASO 1 day after seeding and grown for 6 days before harvest and quantification of the level of RNA. The cells didn’t show any morphological changes or cell death for any of the ASO chimeras or gapmer, targeted or non-targeted. Both designs of LNA-DNA-PNA chimeras showed potent silencing by gymnosis, again comparable to the extent of silencing seen for the LNA gapmer (Figure 7). As was seen for the lipid-based experiments, the difference between the chimeras containing LNA or DNA at the DNAPNA junction was insignificant, suggesting that potency is not limited by affinity in this range.41 LNA gapmers represent a gold standard for potency among ASO designs.39, 43-44 Nevertheless, LNA gapmer ASOs also have some liabilities – including higher toxicity for some sequences.44-48 A new ASO design with the potency of an LNA gapmer but with a reduction in both LNA and PS content could show improved therapeutic properties. PNA-DNA-LNA gapmers may be such a design, matching the potency of LNA gapmers in cultured cells both by lipid transfection and by


Biochemistry

gymnosis. This result justifies in vivo exploration of the potency and toxicity of PNA- and LNAcontaining chimeric ASOs.

HEPA 1-6

100


50

E_MALAT1_Gap_LNA E_MALAT1_Ch_DNA E_MALAT1_Ch_LNA 1 3

1 3 0. 1 0. 1 33 0. 66

1 3 0. 11 0. 3 0. 3 66

1 3 0. 1 0. 1 3 0. 3 66

0

0. 1 0. 1 3 0. 3 66 1 3 0. 11 0. 3 0. 3 66


a

[ASO] (µM)

b

MEF

100


50

E_MALAT1_Gap_LNA E_MALAT1_Ch_DNA E_MALAT1_Ch_LNA 1 3

1 3 0. 1 0. 1 33 0. 66

1 3 0. 1 0. 1 3 0. 3 66

1 3 0. 1 0. 1 3 0. 3 66

0

0. 1 0. 1 3 0. 3 66 1 3 0. 11 0. 3 0. 3 66


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[ASO] (µM) Figure 7. PNA-DNA-LNA chimeras silence Malat1 expression after gymnotic delivery into Hepa 1-6 cells (a) or mouse embryonic fibroblasts (b). RNA was harvested after 6 days, quantified using the QuantiGene 2.0 assay, and normalized to Hprt.

In conclusion, we have identified PNA-DNA chimeras with improved affinity and have shown for the first time that PNA-containing chimeras exert potent RNase H mediated gene silencing in mammalian cells. By controlling the structure (nucleotide conformation and PNA linker length) of


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Biochemistry

the junction between the DNA and PNA portions of this chimeric structure, we increased the affinity of these chimeric ASOs. Nevertheless, this affinity did not translate to higher silencing potency in cells. However, we showed that one LNA wing and one PNA wing could be used to flank a DNA gap, creating ASOs with reduced PS content that combine high affinity and potency with outstanding nuclease stability. Our results, including potent silencing after gymnotic delivery, suggest that these LNA-DNA-PNA chimeras should be advanced into testing in vivo, a context where reducing the PS content of ASOs is a useful strategy to improve the therapeutic index of this promising class of drugs.

AUTHOR INFORMATION Corresponding Authors: *Email: [email protected]; Phone: +1 (774) 455-3784; Fax: +1 (508) 856-6696

ORCID JKW: 0000-0001-5706-1734 AJD: 0000-0003-4707-873X

Notes The authors declare that no competing financial interests exist.

ACKNOWLEDGMENTS This work was supported by the Engineering and Physical Sciences Research Council (EP/M003973/1), the European Commission (Marie Curie Career Integration Grant to JKW), and the Ono Pharma Foundation (Breakthrough Science Award to JKW). We thank Masad Damha (McGill University) and Veenu Aishwarya (AUM Lifetech) for kindly providing 2'F-ANA phosphoramidites.


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SUPPORTING INFORMATION Additional oligonucleotide sequences and characterization (Table S1), synthetic methods and characterization for PNA-G monomer and PNA linkers (Schemes S1-S2); additional CD spectra (Figures S1-S4), additional RNase H assays (Figures S5-S8), details of the PNA synthesis cycle on the Expedite 8909, supporting references.


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Next-generation PNA chimeras exhibit high affinity and potent gene

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