A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides

Abstract

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5-15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

Fig 1. Schematic of the tandem antisense oligonucleotide approach.

WT and Mut mRNA can interact with the gapmer or inhibitor, resulting in the formation of the most favorable thermodynamically duplexes (WT RNA/inhibitor and Mut RNA/gapmer). Possible to form duplexes of RNA variants with inhibitor antisense oligonucleotide (blue) and gapmer antisense oligonucleotide (orange-black) are presented. ASO molecules are designed in a way that gapmer preferentially binds the mutated RNA, promoting its cleavage with RNase H, while inhibitor preferentially hybridize to the wild type RNA, protecting it from RNase H activity.

Fig 2. The RNase H assay results for G291C SNP within SCA3 mRNA.

(A) kinetics of the WT and Mut RNAs cleavage in the presence of the gapmer (fKM) and the shorter (If1) or longer (If2) inhibitor, where black squares indicate degradation of WT RNA in the presence of the If1/fKM/Mut RNA mixture, red dots—degradation of WT RNA in the presence of the If2/fKM/ Mut RNA mixture, blue triangles—degradation of Mut RNA in the presence of the If1/fKM/WT RNA mixture and green triangles—degradation of Mut RNA in the presence of the If2/fKM/WT RNA mixture, (B) stability of the WT RNA (green bar) and Mut RNA (red bar) in the presence of: gapmer fKM only (first pair of bars from the left), gapmer fKM and short inhibitor If1 (second) or gapmer fKM and longer inhibitor If2 (third), and in the WT/Mut RNA/fKM/If1 mixture (fourth) and WT/Mut RNA/fKM/If2 mixture (fifth); statistically significant differences between the mean hydrolysis efficiency of the RNA variants (P<0.05) are marked with asterisk (C,D) Results of HeLa cells cotransfection with WT/Mut G291C-pEGFP constructs and different amounts of inhibitor and gapmer antisense oligonucleotides. qPCR results of tandem approach with (C) shorter inhibitor If1, (D) Longer inhibitor If2. Statistically significant differences between the mean of the RNA variants expression (P<0.05) are marked with asterisk.

Fig 3. The RNase H assay results for C692G SNP within APP mRNA.

(A) kinetics of the WT and Mut RNAs cleavage in the presence of the gapmer (bKM) and the shorter (Ib1) or longer (Ib2) inhibitor, where black squares indicate degradation of WT RNA in the presence of the Ib1/bKM/Mut RNA mixture, red dots—degradation of WT RNA in the presence of the Ib2/bKM/ Mut RNA mixture, blue triangles—degradation of Mut RNA in the presence of the Ib1/bKM/WT RNA mixture and green triangles—degradation of Mut RNA in the presence of the Ib2/bKM/WT RNA mixture, (B) stability of the WT RNA (green bar) and Mut RNA (red bar) in the presence of: gapmer bKM only (first pair of bars from the left), gapmer bKM and short inhibitor Ib1 (second) or gapmer bKM and longer inhibitor Ib2 (third), and in the WT/Mut RNA/bKM/Ib1 mixture (fourth) and WT/Mut RNA/bKM/Ib2 mixture (fifth); statistically significant differences between the mean hydrolysis efficiency of the RNA variants (P<0.05) are marked with asterisk, (C,D) Results of HeLa cells cotransfection with WT/Mut C692G-pEGFP constructs and different amounts of inhibitor and gapmer antisense oligonucleotides. qPCR results of tandem approach with (C) shorter inhibitor Ib1, (D) Longer inhibitor Ib2. Statistically significant differences between the mean of the RNA variants expression (P<0.05) are marked with asterisk.

Fig 4. The RNase H assay results for G46A SNP within SNCA mRNA.

(A) kinetics of the WT and Mut RNAs cleavage in the presence of the gapmer (kKM) and the shorter (Ik1) or longer (Ik2) inhibitor, where black squares indicate degradation of WT RNA in the presence of the Ik1/kKM/Mut RNA mixture, red dots—degradation of WT RNA in the presence of the Ik2/kKM/ Mut RNA mixture, blue triangles—degradation of Mut RNA in the presence of the Ik1/kKM/WT RNA mixture and green triangles—degradation of Mut RNA in the presence of the Ik2/kKM/WT RNA mixture, (B) stability of the WT RNA (green bar) and Mut RNA (red bar) in the presence of: gapmer kKM only (first pair of bars from the left), gapmer kKM and short inhibitor Ik1 (second) or gapmer kKM and longer inhibitor Ik2 (third), and in the WT/Mut RNA/kKM/Ik1 mixture (fourth) and WT/Mut RNA/kKM/Ik2 mixture (fifth). Statistically significant differences between the mean hydrolysis efficiency of the RNA variants (P<0.05) are marked with asterisk, (C,D) Results of HeLa cells cotransfection with WT/Mut G46A -pEGFP constructs and different amounts of inhibitor and gapmer antisense oligonucleotides. qPCR results of tandem approach with (C) shorter inhibitor Ik1, (D) Longer inhibitor Ik2. Statistically significant differences between the mean of the RNA variants expression (P<0.05) are marked with asterisk.

Fig 5. The influence of the shorter inhibitor Ib1 on the cleavage of RNA C692G transversion variants in the presence of bKM gapmer oligonucleotide.

(A) wild type RNA allele C (B) mutated RNA allele G. The sequence and the cleavage sites of analyzed RNAs are presented above gel images. The concentrations of WT and/or Mut RNA variants and the gapmer were 100 nM and 10 nM, respectively. The concentration of the Ib1 inhibitor ranged from 0.01 to 25 μM, as indicated by numbers above the lines. RNase H assay of designed model RNAs usually resulted in 1–2 main products of cleavage and a few minor products. The cleavage pattern is characteristic for particular duplex and could differ depending on presence or absence of a mismatch. Abbreviations in the gel picture mean as follow: T1- RNase T1 cleavage products, F—formamide hydrolysis products, C—control sample. The control sample contained all the mixture components exept for gapmer oligonucleotide.

Fig 6. The RNase H assay results for A693G SNP within APP mRNA.

(A) kinetics of the WT and Mut RNAs cleavage in the presence of the gapmer (eKM) and the shorter (Ie1) or longer (Ie2) inhibitor, where black squares indicate degradation of WT RNA in the presence of the Ie1/eKM/Mut RNA mixture, red dots—degradation of WT RNA in the presence of the Ie2/eKM/ Mut RNA mixture, blue triangles—degradation of Mut RNA in the presence of the Ie1/eKM/WT RNA mixture and green triangles—degradation of Mut RNA in the presence of the Ie2/eKM/WT RNA mixture, (B) stability of the WT RNA (green bar) and Mut RNA (red bar) in the presence of: gapmer eKM only (first pair of bars from the left), gapmer eKM and short inhibitor Ie1 (second) or gapmer eKM and longer inhibitor Ie2 (third), and in the WT/Mut RNA/eKM/Ie1 mixture (fourth) and WT/Mut RNA/eKM/Ie2 mixture (fifth). Statistically significant differences between the mean hydrolysis efficiency of the RNA variants (P<0.05) are marked with asterisk, (C,D) Results of HeLa cells cotransfection with WT/Mut A693G -pEGFP constructs and different amounts of inhibitor and gapmer antisense oligonucleotides. qPCR results of tandem approach with (C) shorter inhibitor Ie1, (D) Longer inhibitor Ie2. Statistically significant differences between the mean of the RNA variants expression (P<0.05) are marked with asterisk.