RESUMO
CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , RNA Bacteriano/química , Clivagem do DNA , DNA Forma A/química , RNA Bacteriano/metabolismo , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Relação Estrutura-AtividadeRESUMO
We report the first syntheses of three nucleoside analogues, namely, 2',4'-diOMe-rU, 2'-OMe,4'-F-rU, and 2'-F,4'-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4'-α-position of a 4',5'-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2',4'-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4'-α-F/OMe substituents reinforce the C3'-endo ( north) conformation of 2'-OMe-rU. Furthermore, the predominant C2'-endo ( south/ east) conformation of 2'-F-araU switches to C3'-endo upon introduction of these substituents at C4'. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4'-substituents can modulate the binding affinity of the parent 2'-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics.
RESUMO
Sugar puckering of nucleosides impacts nucleic acid structures; hence their biological function. Similarly, nucleoside-based therapeutics may adopt different conformations affecting their binding affinity, DNA incorporation, and excision rates. As a result, significant efforts have been made to develop nucleoside analogues adopting specific conformations to improve bioactivity and pharmacokinetic profiles of the corresponding nucleoside-containing drugs. Understanding and ultimately predicting these conformational preferences would significantly help in the design of more effective structures. We report herein a computational study based on hybrid QM/MM umbrella sampling simulations that allow the accurate prediction of the sugar conformational preferences of chemically modified nucleosides in solution. Moreover, we pair these simulations with natural bond orbital (NBO) analysis to gain key insights into the role of substituents in the conformational preferences of these nucleosides.
Assuntos
DNA/química , Teoria Quântica , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'ß) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and ß epimers could be obtained by a nonstereoselective approach starting from 2'-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the ß-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.
Assuntos
Inativação Gênica , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Ribonucleotídeos/genética , Termodinâmica , Uridina/química , Uridina/metabolismoRESUMO
Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) - nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior - have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.
RESUMO
In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.
Assuntos
Proteínas Argonautas/genética , Inativação Gênica , RNA Interferente Pequeno/genética , Açúcares/química , Proteínas Argonautas/antagonistas & inibidores , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla , RNA Interferente Pequeno/antagonistas & inibidores , Uridina/químicaRESUMO
Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC invasion. We developed antibody-antisense oligonucleotide (AON) conjugates to target and reduce DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the GSCs, such as CD44 and EphA2, conjugated to chemically modified AONs against DRR/FAM107A, which were designed as chimeras of DNA and 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize, accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first example of an antibody-antisense strategy against cancer stem cells.