WO2025043118A1

WO2025043118A1 - Making tiny rnas inside the cell

Info

Publication number: WO2025043118A1
Application number: PCT/US2024/043488
Authority: WO
Inventors: Kotaro NAKANISHI
Original assignee: Ohio State Innovation Foundation
Current assignee: Ohio State Innovation Foundation
Priority date: 2023-08-23
Filing date: 2024-08-22
Publication date: 2025-02-27
Anticipated expiration: 2026-02-23

Abstract

Guide RNA can be used with an Argonaute (AGO) molecule, wherein said AGO molecule, when loaded with said guide RNA, cleaves a target nucleic acid. This guide RNA can be a cityRNA. Described herein are cityRNAs which have been modified with a Booster nucleic acid to increase their ability to cleave target nucleic acid through AGO.

Description

MAKING TINY RNAS INSIDE THE CELL

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/578,265, filed August 23, 2023, and U.S. Provisional Patent Application No. 63/623,530, filed January 22, 2024, both entitled “MAKING TINY RNAS INSIDE THE CELL,” which are incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government Support under grant/contract number R01 GM138997 awarded by the National Institutes of Health. The Government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on August 22, 2024, as an .XML file entitled “103361- 573WO1_ST26” created on August 19, 2024, and having a file size of 256,145 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD

The present disclosure provides guide RNA (gRNA) compositions and complexes comprising a tinyRNA (tyRNA) and/or cleavage-induced tinyRNA (cityRNA) hybridized to a Booster nucleic and methods of use thereof.

BACKGROUND

MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression post- transcriptionally (Kozomara 2019; Bartel 2018). Their sequences differ, but their lengths generally fall within a range of 20-23 nucleotides because the precursor miRNAs are processed by Dicer, which is a molecular ruler that generates size-specific miRNA duplexes (Zhang 2004; Macrae 2006). After those duplexes are loaded into Argonaute proteins (AGOs), one of the two strands is ejected while the remaining strand (guide strand) and the AGO form the RNA-induced silencing complex (RISC) (Nakanishi 2016). Therefore, the 20-23 -nucleotide length is the hallmark of intact miRNAs. This size definition has been exploited as the rationale for eliminating -18 nucleotide RNAs when AGO-bound miRNAs are analyzed by next-generation RNA sequencing (RNAseq). However, RNAseq without a size exclusion reported a substantial number of ~18-nucleotide RNAs bound to AGOs (Kuscu 2018; Gangras 2018; Kumar 2014). Such tiny gRNAs (tyRNAs) are known to be abundant in extracellular vesicles of plants (Baldrich 2019), but little was previously known about their roles or biogenesis pathways. In mammals, the roles of tyRNAs have been even more enigmatic.

In 2004, two groups reported that among the four human AGO paralogs, only AGO2 showed the guide-dependent target cleavage in vitro (Liu 2004; Meister 2004). Since then, AGO1, AGO3, and AGO4 were thought to be deficient in RNA cleavage, even though AGO3 shares the same catalytic tetrad with AGO2. Recently, it was revealed that specific miRNAs such as 23- nucleotide miR-20a make AGO3 a slicer, but the activity was much lower than that of AGO2 (Park 2017).

What is needed in the art are gRNA to be used with AGO complexes, as well as structures which can increase the efficacy, binding, or efficiency of the gRNA: AGO complex.

SUMMARY

The present disclosure provides gRNA compositions and complexes comprising a tyRNA and/or cityRNA hybridized to a Booster nucleic and methods of use thereof.

In one aspect, disclosed herein is a gRNA comprising a cityRNA hybridized with a Booster nucleic acid, wherein the Booster nucleic acid comprises a tetranucleotide loop or a 3 ' 2-nucleotide overhang.

In one aspect, disclosed herein is a cityRISC complex comprising a gRNA and an AGO, wherein the gRNA comprises a cityRNA hybridized to a Booster nucleic acid, and wherein the Booster nucleic acid comprises a tetranucleotide loop or a 3' 2-nucleotide overhang.

In some embodiments, the cityRNA is 16 nucleotides or less in length. In some embodiments, the cityRNA is 14 nucleotides or less in length. In some embodiments, the Booster nucleic acid hybridizes with the cityRNA. In some embodiments, the Booster nucleic acid and cityRNA together form a secondary structure. In some embodiments, the gRNA is greater than 18 nucleotides in length when Booster is hybridized to cityRNA. In some embodiments, the Booster nucleic acid is RNA or DNA. In some embodiments, the Booster nucleic acid comprises at least 2 nucleotides. In some embodiments, the Booster nucleic acid comprises 25-38 nucleotides.

In one aspect, disclosed herein is a cell comprising the gRNA of any preceding aspect or the cityRISC complex of any preceding aspect. In one aspect, disclosed herein is a method of regulating expression of a target nucleic acid using a cityRISC complex, wherein the cityRISC complex comprises an AGO protein and a gRNA, wherein said gRNA comprises a cityRNA hybridized with a Booster nucleic acid.

In one aspect, disclosed herein is a method of determining a suitable Booster nucleic acid, the method comprising identifying a cityRNA, hybridizing said cityRNA to a Booster nucleic acid, detecting whether the cityRNA hybridized with the Booster nucleic acid is more efficient at loading the cityRNA into an AGO or regulating gene expression, and selecting the suitable Booster nucleic acid that loads the cityRNA into the AGO and regulating gene expression.

In some embodiments, the AGO molecule comprises Argonautel (AG01), Argonaute2 (AG02), Argonaute3 (AG03), or Argonaute4 (AG04). In some embodiments, the target nucleic acid is RNA or DNA.

In some embodiments, the method of any preceding aspect comprises the cityRNA comprising 16 nucleotides or less in length. In some embodiments, the method of any preceding aspect comprises the cityRNA comprising 14 nucleotides or less in length. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid hybridizing with the cityRNA. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid and cityRNA together forming a secondary structure.

In some embodiments, the method of any preceding aspect comprises the gRNA having greater than 18 nucleotides in length when Booster is hybridized to cityRNA. In some embodiments, the method of any preceding aspect comprises a Booster RNA or a Booster DNA. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid comprising at least 2 nucleotides. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid comprising 25-38 nucleotides.

In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid protecting the cityRNA from degradation by a nuclease. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid improving binding the cityRNA to the AGO.

BRIEF DESCRIPTION OF FIGURES

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

Figures 1A and IB show the structure of target-bound AGO3-cityRNA. Figure 1A shows the crystal structure of AGO3 in complex with 14-nt miR-20a and 16-nt target RNA. The structure of AGO3 is depicted as ribbon model. The cityRNA guide and target are colored in red and blue, respectively. The sequence alignment between the guide and target is shown. The hydrogen bonds observed in the crystal structure are shown as black lines. Figure IB shows the superposition of the current structure (blue) with the AG02 structure in State III (pink) (PDB ID: 6N4O). For clarity, neither the guide nor the target is shown.

Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 21 show the in vitro target cleavage by the homogeneous AGO3-cityRISCs. Figure 2A and 2B show the cleavage of different-length targets (Figure 2A) by the homogeneous AGO3: 14-nt miR-20a. Target cleavage with [target] < [RISC], (Figure 2B) Initial velocities, vo, for fully complementary targets, were determined by fitting the data to a single exponential, vo was determined by three independent experiments, [target] = 2.5 nM. [RISC] = 10 nM. Figures 2C and 2D show the cleavage of different-length targets by the homogeneous AGO3: 14-nt let-7a. Figures 2E and 2F show the cleavage of chimeric targets by the homogeneous AGO3: 14-nt miR-20a. Figures 2G and 2H show the cleavage of chimeric targets by the homogeneous AGO3: 14-nt let-7a. Figure 21 shows the schematic of target recognition by city RISC.

Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 31, 3J, 3K, and 3L show the in vitro target cleavage by the homogeneous AGO2-mature RISCs and -cityRISCs. Figures 3A and 3B show the cleavage of different-length targets (Figure 3A) by the homogeneous AGO2:23-nt miR-20a. Target cleavage with [target] < [RISC], Figure 3B shows the initial velocities, vo, for fully complementary targets, were determined by fitting the data to a single exponential, vo was determined by three independent experiments, [target] = 2.5 nM. [RISC] = 10 nM. Figure 3C and 3D show the cleavage of different-length targets by the homogeneous AGO2:21-nt let-7a. Figure 3E and 3F show the cleavage of different-length targets by the homogeneous AGO2: 14-nt miR- 20a. Figures 3G and 3H show the cleavage of different-length targets by the homogeneous AGO2: 14-nt let-7a. Figure 31 and 3 J show the cleavage of chimeric targets by the homogeneous AGO2: 14-nt miR-20a. Figures 3K and 3L show the cleavage of chimeric targets by the homogeneous AGO2: 14-nt let-7a.

Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41 and 4J show gene silencing by cityRNAs. Figure 4A shows the schematics of the 7a-7a target site recognized by 21-nt let-7a (top) and 14- nt let-7am (or 14-nt let-7a) (bottom). The tl5-t21 of the let-7a-binding site is colored in blue. Figure 4B shows the DLR assays in HEK293T cells co-transfected with psiCHECK-7a-7a and either of the designated RNAs. Figure 4C shows the schematics of the 7a-20a target site recognized by 21-nt let-7a (top) and 14-nt let-7am (or 14-nt let-7a) (bottom). The tl 5-t23 of the miR-20a-binding site is colored in orange. Figure 4D and 4E show the DLR assays in HEK293T cells co-transfected with psiCHECK-7a-20a and either of the designated RNAs. Figures 4F and 4G show the DLR assays in HC116 AGOl/2(-/-) cells co-transfected with psiCHECK-7a-20a and either of the designated RNAs. Figures 4H, 41, and 4J show the silencing activities of 21 -nt let- 7a duplex and cyBR-7am against the 7a-7a and 7a-20a reporters in HCT116 AG01/2(-/-) (Figure 4H), HCT116 wild-type (Figure 41), and HEK293T cells (Figure 4J).

Figures 5A, 5B, 5C, and 5D show the conformations of miRNA-associated AG02 and tyRNA-associated AG03. Figure 5 A shows an F₀-F_c omit map of the guide and target strand (3 c) show a continuous electron density map of the gl-g8, while the guide after g8 has no F₀-F_c omit map. A polder map of each g9, glO, gl l, and either of gl2, gl3, or gl4 is shown together. Figure 5B shows a denaturing gel image of the co-crystallized target RNA. Figure 5C shows the superposition of four crystal structures of AG02 in State I (PDB ID: 40LA), State II (PDB ID: 4W5O), State III (PDB ID: 6N4O), and State IV (PDB ID: 6MDZ). For clarity, neither the guide nor the target is shown. Figure 5D shows the pairing of the guide (red) and target (blue) bound to AG02 (States II-IV). For clarity, no protein is shown.

Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 61, 6J, 6K, 6L, 6M, and 6N show that the sequence of cityRNA is the primary factor of target cleavage by cityRISC. Figure 6A shows the singleturnover kinetics of target cleavage by the homogeneous AGO3-RISC with 14-nt miR-20a, let- 7a, miR-19b, or miR-16. Each RISC was incubated with 60-, 58-, 60-, or 59-nt corresponding target with a 5'-cap radiolabeling. Target cleavage by the AG03 : 14-nt miR-16 was not detectable (ND). Figures 6B, 6C, and 6D show the cleavage of different-length targets (Figure 6B) by the homogeneous AG03: 14-nt miR-19b. Target cleavage with [target] < [RISC], [target] = 2.5 nM. [RISC] = 10 nM. Figure 6C shows the reaction products were run on a denaturing gel. Figure 6D shows the initial velocities, vO, for fully complementary targets were determined by fitting the data to a single exponential. vO was determined by three independent experiments. Figures 6E and 6F show the cleavage of different-length targets (Figure 6E) by the homogeneous AGO3: 14-nt miR-16. Target cleavage with [target] < [RISC], [target] = 2.5 nM. [RISC] = 10 nM. Figure 6F shows the reaction products were run on a denaturing gel. No target cleavage was detected. Figure 6G shows the models of target cleavage by AGO3-cityRISC. In Figure 6G, when AGO3-cityRISC uses the seed (g2-g8) of the cityRNA (red) to recognize a target (blue), the t2-t8 is fixed while the rest of the target is flexible in solvent. The g9-gl4 is also free to move within the nucleic acidbinding channel. When thel5-t23 has a specific sequence, it works as a “Target-cleavage Enhancer in Cis” (TEC) and is recognized by the AGO3 -cityRISC, which helps the t9-t 14 is basepaired with the g9-gl4. As a result, the AGO3-cityRISC cleaves the target between tlO and tl 1. Figures 6H, 61, and 6J show the cleavage of the chimeric targets (Figure 6H) by the homogeneous AGO3: 14-nt miR-19b. Target cleavage with [target] < [RISC], [target] = 2.5 nM. [RISC] = 10 nM. Figure 61 shows the reaction products were run on a denaturing gel. Figure 6J shows the initial velocities, vo, for fully complementary targets were determined by fitting the data to a single exponential, vo was determined by three independent experiments. Figure 6K, and 6L show the cleavage of the chimeric targets (Figure 6J) by the homogeneous AGO3: 14-nt miR-16. Target cleavage with [target] < [RISC], [target] = 2.5 nM. [RISC] = 10 nM. Figure 6L shows the reaction products were run on a denaturing gel. No target cleavage was detected. Figure 6M, and 6N show the cleavage of the chimeric targets (Figure 6M) by the homogeneous AGO2:21-nt let-7a. Target cleavage with [target] < [RISC], [target] = 2.5 nM. [RISC] = 10 nM. Figure 6N shows the initial velocities, vo, for fully complementary targets were determined by fitting the data to a single exponential, vo was determined by three independent experiments.

Figures 7A, 7B, 7C, 7D, 7E, and 7F show the AGO2-mature RISC do not use a TEC for target recognition. Figure 7A shows the single-turnover kinetics of target cleavage by the homogeneous AGO2-RISC with 14- and 21-nt let-7a against a 58-nt target. Target cleavage with [target] < [RISC], [target] = 2.5 nM. [RISC] = 10 nM. Initial velocities, vo, for fully complementary targets were determined by fitting the data to a single exponential, vo was determined by three independent experiments. Figure 7B shows the single-turnover kinetics of target cleavage by the homogeneous AGO2-RISC with 14- and 23 -nt miR-20a against a 60-nt target. Figures 7C and 7D show the cleavage of the chimeric targets (Figure 7C) by the homogeneous AGO3 :23-nt miR-20a. Figures 7E and 7F show the cleavage of the chimeric targets (Figure 7E) by the homogeneous AGO3:21-nt let-7a.

Figures 8A, 8B, 8C, 8D, 8E, and 8F show the gRNAs used for DLR assays. Figure 8A shows the structures of RNAs used for DLR assays. Circle dots indicate 5' monophosphate group. Figure 8B shows the native PAGE analysis of the RNAs used for DLR assays. Left: the original image. Right: high contrast image. Figure 8C shows the DLR assays in HCT116 wile-type, A549, and HeLa cells co-transfected with psiCHECK-7a-20a and either 21-nt let-7a duplex or cyBR- 7a^m. Figures 8D and 8E show the DLR assays in HEK293T, HCT116 wile-type, and HCT116 AG01/2(-/-) cells co-transfected with psiCHECK-20a-20a and either 21-nt let-7a duplex or cyBR- 7a^m. 14-nt let-7a^m is not complementary to the 20a-20a target site (Figure 8D). Figure 8F shows the DLR assays in HCT116 wile-type, A549, HeLa cells co-transfected with psiCHECK-7a-20a and either cyDR-7a^m or cyDR-7a.

Figure 9 shows an example of a Booster.

Figure 10 shows another example of a Booster. Figure 11 shows Booster helps 14-nt cityRNAs to be loaded into endogenous AGOs even at 37 °C. The gene silencing activity of different RNAs was evaluated by Dual -Luciferase Reporter assay with (left) and without (right) RT incubation.

Figure 12 shows that Boosters help 14-nt cityRNAs to be loaded into endogenous AGOs and that the tl 5-t23 of the 23-nt miR-20a target enhances the target cleavage. The gene silencing activity of different RNAs was evaluated by Dual-Luciferase Reporter assay.

Figure 13 shows In vitro target cleavage of the “7a-20a,” “7a- 16,” “7a- 19b,” and “7a-7a” by AG02 programmed with 14-nt let-7a (left) and 21-nt let-7a (right). For example, the tl -tl4 and the tl 5-t21 of the “7a-20a” target have sequences complementary to the gl-gl4 and the gl5-g21 of 21-nt let-7a, respectively.

Figure 14 shows dual-Luciferase Reporter assay. LNA: Locked Nucleic Acid.

Figures 15 A, 15B, 15C, 15D, 15E, 15F, 15G, and 15H show unpaired target region upstream tyRNA-binding site enhances target cleavage by cityRISCs. Figures 15A and 15B show the single-turnover kinetics of target cleavage by the homogeneous AG03-RISC loaded with 14- nt miR-20a, let-7a, miR-19b, or miR-16. Each RISC was incubated with the 60-, 58-, 60-, or 59- nt corresponding target with a 5'-cap radiolabel. Target cleavage by the AG03 : 14-nt miR-16 was not detectable (ND). Figures 15C, 15D, 15E, 15F, 15G, and 15H show the single-turnover cleavage assays of different-length targets by homogeneous AG03: 14-nt miR-20a (Figures 15C and 15D), AG03: 14-nt let-7a (Figure 15E and 15F), AG02: 14-nt miR-20a (Figure 15G), and AG02: 14-nt let-7a (Figure 15H). Target RNA lengths listed do not include the two adenylates at the 3' end (grey). Initial velocities, vo, were determined by fitting the data to a single exponential with three independent experiments. For all cleavage assays in this figure, [target] = 2.5 nM. [RISC] = 10 nM. Data are mean ± SD.

Figures 16A, 16B, 16C, 16D, 16E, and 16F show the in vitro chimeric target cleavage by homogeneous AGO3- and AGO2-cityRISCs. Figures 16A, 16B, and 16C show the singleturnover cleavage of the 20a^B-based chimeric targets (Figure 16A) by homogeneous AGO3 : 14-nt miR-20a (Figure 16B) and AGO2: 14-nt miR-20a (Figure 16C). Figure 16D, 16E, and 16F show the single-turnover cleavage of the 7a^B-based chimeric targets (Figure 16D) by homogeneous AGO3: 14-nt let-7a (Figure 16E) and AGO2: 14-nt let-7a (Figure 16F). [target] = 2.5 nM. [RISC] = 10 nM. The assays were triplicated. Data are mean ± SD.

Figures 17A, 17B, 17C, 17D, 17E, 17F, and 17G show the mature RISC and cityRISC have different target preferences for cleavage. Figure 17A shows that the AGO2-RISC changes the preferred target site with the conversion from mature miRNA to cityRNA. Figures 17B, 17C, and 17D show the relative vo, Acat, and K_m of the 7a^B-20a^T cleavage to that of the 7a^B-7a^T by AGO2:21-nt let-7a (Figure 17B), AG02: 14-nt let-7a (Figure 17C), and AG03: 14-nt let-7a (Figure 17D). Figures 17E and 17F show the relative vo, feat, and K_m of the 20a^B-20a^T cleavage to that of the 20a^B-7a^T by AGO2: 14-nt miR-20a (Figure 17E) and AGO3: 14-nt miR-20a (Figure 17F). Figure 17G shows the model mechanisms of target cleavage by cityRISC: The 20a^T-like TAM (blue) is not tightly recognized by the TAM-recognition site (green) on AGO. The resultant dynamic lets the target quickly form a duplex with the cityRNA, thereby drastically facilitating the catalytic reaction (top). A 7a^T-like TAM (orange) is recognized by the TAM-recognition site, which reduces the chance of the target base pairing with the g9-gl4 and thus moderately facilitates the catalytic reaction (bottom). The assays were triplicated. Data are mean ± SD.

Figures 18 A, 18B, 18C, 18D, 18E, 18F, 18G, 18H, 181, 18 J, 18K, and 18L show the gene silencing by cityRNAs. Figure 18A shows the DLR assays in HEK293T: co-transfected with psiCHECK-7a^B-7a^T and designated RNA. cyBR-7a^m is composed of 14-nt modified let-7a (red) and Booster (grey). Figure 18B shows the DLR assays in HEK293T: co-transfected with psiCHECK-7a^B-20a^T and designated RNA. Figure 18C shows the DLR assays in HCT116, A549, and HeLa: co-transfected with psiCHECK-7a^B-20a^T and 21 -nt let-7a duplex or cyBR-7a^m. Figure 18D and 18E show the DLR assays in HEK293T (Figure 18D), HCT116, A549, and HeLa (Figure 18E): co-transfected with psiCHECK-7a^B-20a^T and cyDR-7a^m or cyDR-7a. Figures 18F and 18G show the endogenous immunofluorescence staining of FLAG-AG02 (Figure 18F), -AG03 (Figure 18G), and co-localization with 14-nt Cy 3 -conjugated let-7a in HeLa. Figures 18H, 181, and 18J show the comparison of silencing abilities between 14- and 21-nt let-7a (Figure 18H), and 14- and 22-nt miR-92a (Figures 181 and 18J) across four cell lines. Figures 18K and 18L shows the reliance of cityRISC-driven gene silencing on target cleavage. DLR assays in four cell lines co-transfected with psiCHECK-7a^B-7a^T (orange) (Figure 18K) or -7a^B-20a^T (blue) (Figure 18L), including a fully complementary (solid) or tlO-tl l mismatched (empty) tyRNA-binding site, and an siRNA duplex, cyBR-7a^m, or cyDR-7a^m. (Figures 18 A, 18B, 18C, 18D, and 18E, Figures 18K and 18L) Data shown as relative luciferase activity and normalized to no-guide control, (Figures 18H, 181, and 18J) data shown as (gene silencing %) = (1 - relative luciferase activity) x 100%. All assays were triplicated. Data are mean ± SD. *P < 0.05; **P < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (ANOVA with Dunnett’s post-hoc test).

Figures 19A, 19B, 19C, 19D, 19E, 19F, and 19G show the conformations of AGO3- cityRISC and AGO2-mature RISC. Figure 19A shows the SDS-PAGE analysis of purified homogeneous AGO3: 14-nt miR-20a. Figure 19B shows the nucleotide modifications added to target RNAs to avoid cleavage by cityRISC. The phosphorothionate group and nucleotide with 2'- OMe are colored red and green, respectively. The 5 '-end radiolabeled monophosphate group is depicted as a yellow circle. 14-nt miR-20a is shown in red. Figure 19C shows the in vitro target cleavage of the unmodified and modified targets by homogeneous AG03: 14-nt miR-20a. Figure 19D shows an F₀-Fc omit map of the guide and target strand (3 c) show a continuous electron density map of the gl-g8, while the guide after g8 has no F₀-Fc omit map. Polder maps (4 c) of g9, glO, gl 1, either gl 3 or gl4, and maps of t9 and tlO are shown together. Figure 19E shows a denaturing gel image of the co-crystallized target RNA. Figure 19F shows the pairing of the guide (red) and target (blue) bound to AG02 (States II-IV). For clarity, no protein is shown. Figure 19G shows the superposition of four crystal structures of AG02-mature RISC in State I (PDB ID: 40LA), State II (PDB ID: 4W5O), State III (PDB ID: 6N4O), and State IV (PDB ID: 6MDZ) on their PIWI domains.

Figures 20A, 20B, 20C, 20D, 20E, 20F, 20G, 20H, 201, 20 , and 20K show the in vitro target cleavage by non-cityRNAs and mature RISCs. Figure 20A shows the base pairing of 14-nt miR-19b (red) with 14- and 23 -nt miR-19b targets (green). Figures 20B and 20C show the representative denaturing gels of 23- or 14-nt target cleavage by homogeneous AGO3 : 14-nt miR- 19b (Figure 20B) or AGO2: 14-nt miR-19b (Figure 20C). Cleavage product is plotted as a function of time (bottom). Figure 20D shows the base pairing of 14-nt miR-16 (red) with 14- and 22-nt miR-16 targets (purple). Figures 20E and 20F show the representative denaturing gels of 22- or 14-nt target cleavage by homogeneous AGO3: 14-nt miR-16 (Figure 20E), or AGO2: 14-nt miR- 16 (Figure 20F). Cleavage product is plotted as a function of time (bottom). Figure 20G shows the binding isotherms of the indicated four tyRNA-associated RISCs with targets whose sequence is fully complementary to their parental miRNA. Figure 20H shows the base pairing of 23-nt miR- 20a (red) with 14-, 16-, 18-, 20-, and 23-nt complementary targets (blue). Figure 201 shows the time course of different-length target cleavage by homogeneous AGO2:23-nt miR-20a. Figure 20 shows the base pairing of 21-nt let-7a (red) with 14-, 16-, 18-, 20-, and 21-nt complementary targets (orange). Figure 20K shows the time course of different-length target cleavage by homogeneous AGO2:21-nt let-7a. For all cleavage assays in this figure, [target] = 2.5 nM. [RISC] = 10 nM. Target RNA lengths do not include the two 3' end adenylates (grey). The assays were triplicated. Data are mean ± SD.

Figures 21A and 21B show the in vitro target cleavage by cityRISCs and mature RISCs. Figure 21A shows the top: Base pairing of a 58-nt target (black) with 21- and 14-nt let-7a (red). Bottom: Time course of 58-nt target cleavage by homogeneous AGO2 loaded with 21-nt let-7a (red) or 14-nt let-7a (pink). Figure 21B shows the top: Base pairing of a 60-nt target (black) with 21- and 14-nt miR-20a (red). Bottom: Time course of 60-nt target cleavage by homogeneous AG02 with 23-nt miR-20a (red) or 14-nt miR-20a (pink). For all cleavage assays in this figure, [target] = 2.5 nM. [RISC] = 10 nM. The assays were triplicated. Data are mean ± SD.

Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 221, 22J, 22K, and 22L show the in vitro target cleavage by non-cityRISCs and mature RISCs. Figure 22A shows the base pairing of 14-nt miR-19b (red) with 19b^B-based chimeric targets. All targets share the same tl-tl4 complementary to 14-nt miR-19b. Figures 22B and 22C show the representative denaturing gels for cleavage of the chimeric targets by homogeneous AGO3: 14-nt miR-19b (Figure 22B) and AGO2: 14-nt miR-19b (Figure 22C). Time course of chimeric target cleavage by homogeneous AGO2: 14-nt miR-19b (bottom). Figure 22D shows the base pairing of 14-nt miR-16 (red) with 16^B-based chimeric targets. All targets share the same tl-tl4 complementary to 14-nt miR-16. Figures 22E and 22F show the representative denaturing gels for cleavage of the chimeric targets by homogeneous AGO3 : 14-nt miR-16 (Figure 22E) and AGO2: 14-nt miR-16 (Figure 22F). Time course of chimeric target cleavage by homogeneous AGO2: 14-nt miR-16 (bottom). Figure 22G shows the base pairing of 23-nt miR-20a (red) with 20a^B-based chimeric targets. All targets share the same tl -tl 4 complementary to 14-nt miR-20a. Figure 22H shows the time course of chimeric target cleavage by homogeneous AGO2:23-nt miR-20a. Figure 221 shows the base pairing of 21- nt let-7a (red) with 7a^B-based chimeric targets. All targets share the same tl -tl4 complementary to 14-nt let-7a. Figure 22J shows the time course of chimeric target cleavage by homogeneous AGO2:21-nt let-7a. Figures 22K and 22L show the differences in the recognition of 5' upstream flanking region between mature RISC (Figure 22K) and cityRISC (Figure 22L). For all cleavage assays in this figure, [target] = 2.5 nM. [RISC] = 10 nM.

Figures 23A, 23B, 23C, 23D, 23E, 23F, 23G, 23H, 231, 23J, 23K, and 23L show the multiple-turnover kinetics data. Figures 23 A, 23B, 23C, 23D, 23E, and 23F show the Michaelis- Menten plots of AGO2-mature RISC (Figures 23A and 23B) and -cityRISCs (Figures 23C, 23D, 23E, and 23F). Figures 23G, 23H, 231, 23J, 23K, and 23L show the Michaelis-Menten plots of AGO3-mature RISC (Figures 23G and 23H) and -cityRISCs (Figures 231, 23J, 23K, and 23L). The assays were triplicated. Data are mean ± SD. ND, not detectable. [RISC] = 10 nM.

Figures 24A, 24B, 24C, and 24D show the design of cyBR and cyDR and in-cell RISC assembly assay. Figure 24A shows the structures of RNAs used for DLR assays. The strands eventually loaded into AGOs are colored in red. Passengers and Boosters are shown in grey. Strands in black were used as markers in (Figure 24B) Circle dots indicate 5' monophosphate group. Figure 24B shows the native PAGE analysis of the RNAs used for DLR assays (stained with SYBR Gold). Figure 24C shows the schematics of the four base pairing patterns between the two guides (14- and 21-nt let-7a) and the two targets (7a^B-7a^Tand 7a^B-20a^T) used in DLR assays. Figure 24D shows the representative denaturing gel images of in-cell RISC assembly assay.

Figures 25A, 25B, 25C, 25D, 25E, and 25F show the gene silencing by different tyRNAs. Figures 25A, 25B, and 25C show the DLR assays in HEK293T, HCT116, A549, and HeLa cells transfected with cyBR-7a^m (Figure 25A), cyBR-20a^m (Figure 25B), or cyBR-92a^m (Figure 25C) with the indicated psiCHECK-2 plasmid. These cyBRs carry a cityRNA, 14-nt let-7a, miR-20a, or miR-92a. Figure 25D shows the DLR assays in the indicated four cells transfected with cyBR- 7a^m and psiCHECK-20a^B-20a^T. 14-nt let-7a is not complementary to the 20a^B-20a^T target site. Figures 25E and 25F show the DLR assays in the indicated four cells transfected with cyBR-16^m (Figure 25E) or cyBR-19b^m (Figure 25F) with the designated psiCHECK plasmid. These cyBRs carry a non-cityRNA, 14-nt miR-16 or miR-19b.

Figure 26 shows gene silencing by cyDR-92am. DLR assays in HEK293T, HCT116, A549, and HeLa cells co-transfected with psiCHECK-92aB-92aT (left) or psiCHECK-92aB-20aT (right) together with cyDR-92am. Data were shown as relative luciferase activity and normalized to a no-guide control. The assays were triplicated. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001; ns, not significant (ANOVA with Dunnett’s post-hoc test).

Figure 27 shows a schematic of confocal microscopy study to localize cityRISCs. 14-nt let-7a (black strand) conjugated with a Cy3 (yellow circle) at its 3' end was annealed with Delooped Booster (grey) to reconstitute a cyDR, followed by co-transfection with pCAGEN-FLAG- AGO2 into HeLa cells. FLAG-AGO2 was detected with an anti-FLAG antibody, followed by immunofluorescence staining with a secondary antibody conjugated with Alexa Fluor 647 (AF647, red circle). As a result, 14-nt let-7a-associated FLAG-AGO2 is detected as an orange dot, while 14-nt let-7a-associated endogenous AGOs and free cyDR-7a are seen in yellow. 14-nt let- 7a-associated FLAG-AGO3 was detected in the same manner but by transfecting pCAGEN- FLAG-AGO3, instead of pCAGEN-FLAG-AGO2.

Figures 28A and 28B show tlO-tl l mismatches ruin the target cleavage by cityRISCs. Figure 28 A shows the guide and target RNAs used in (Figure 28B). The tlO-tl 1 mismatches are colored black. Figure 28B shows the homogeneous AGO3: 14-nt let-7a, AGO2: 14-nt let-7a, or AGO2:21-nt let-7a was incubated with the 7a^B-7a^T, 7a^B-20a^T, or their corresponding tlO-tl l mismatched targets for 0.5, 20, and 40 min. The reaction was resolved on a denaturing gel.

DETAILED DESCRIPTION

The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

The following definitions are provided for the full understanding of terms used in this specification.

The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.

By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms. As used herein, the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

As used herein, by a “subject” means an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may be prepared by the phosphorami dite method described by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes. A single-stranded oligonucleotide can exist as a linear molecule without any hydrogen-bonded nucleotides, or can fold three-dimensionally to form hydrogen bonds between individual nucleotides along the single stranded oligonucleotide.

The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers. Polynucleotides can be any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. In some embodiments, the polynucleotide is composed of nucleotide monomers of generally greater than 100 nucleotides in length and up to about 8,000 or more nucleotides in length.

The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

The term “complementary” or “complementarity” refers to the topological compatibility or matching together of interacting surfaces of two molecules (e.g., a probe molecule and its target, particularly a DNA guide molecule and a target RNA molecule). Thus, the two molecules (e.g., target and its probe) can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. In the case of nucleotides or polynucleotides (e.g., DNA or RNA), the two molecules are complementary if they have sufficiently compatible nucleotide base-pairs such that the two molecules can hybridize. The term “complementary,” as it relates to nucleotide molecules (e.g., nucleotides, oligonucleotides, polynucleotides, modified nucleotides, etc.), is intended to include two or more nucleotide molecules which have 100% complementarity (e.g., each nucleotide in a sequence of one molecule is the nucleotide base-pair complement of an adjacent nucleotide in a sequence of the second molecule, in sequential order) as well as two or more nucleotide molecules which have less than 100% complementarity but which hybridize under the conditions of the methods disclosed herein.

The term “hybridization” or “hybridizes” refers to a process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex.

The term “anneal” refers to the process by which a single-stranded nucleic acid sequence pairs by hydrogen bonds to a complementary sequence, forming a double-stranded nucleic acid sequence, including the reformation (renaturation) of complementary strands that were separated by heat (thermally denatured). The term “melting” refers to the denaturation of a double-stranded nucleic acid sequence due to high temperatures, resulting in the separation of the double strand into two single strands by breaking the hydrogen bonds between the strands.

The term “target” refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species.

The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e., a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide). One of skill will recognize that nucleic acids (e.g. polynucleotides) can be manipulated in many ways and are not limited to the examples above.

The term “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g. polynucleotide) may include a terminator that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. In some embodiments, the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g. polynucleotide) and a terminator operably linked to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. In some embodiments, the expression cassette comprises an endogenous promoter. In some embodiments, the expression cassette comprises an endogenous terminator. In some embodiments, the expression cassette comprises a synthetic (or non-natural) promoter. In some embodiments, the expression cassette comprises a synthetic (or non-natural) terminator.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, preferably less than about 0.01.

The phrase “codon optimized” as it refers to genes or coding regions of nucleic acid molecules for the transformation of various hosts, refers to the alteration of codons in the gene or coding regions of polynucleic acid molecules to reflect the typical codon usage of a selected organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that selected organism.

Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

The term "nucleobase" refers to the part of a nucleotide that bears the Watson/Crick basepairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.

A polynucleotide sequence is “heterologous” to a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from naturally occurring allelic variants.

The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence with a higher affinity, e.g., under more stringent conditions, than to other nucleotide sequences (e.g., total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5*SSC, and 1% SDS, incubating at 42° C., or, 5*SSC, 1% SDS, incubating at 65° C., with wash in 0.2* SSC, and 0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1 *SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Polypeptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

Guide RNA and other nucleic acids

More than 2,000 microRNAs (miRNAs) have been reported as of 2019 in humans (Kozomora 2018). miRNAs are varied in sequence, but their lengths fall within a range of 19-23 nucleotides (nt) because precursor miRNAs are processed by Dicer which is a molecular ruler that generates size-specific miRNA duplexes (Zhang 2004; Macrae 2006; MacRae 2007). After those duplexes are loaded into AGOs, one of the two strands is ejected while the remaining strand (guide strand) and AGO form the RNA-induced silencing complex (RISC) (Nakanishi 2016; Meister 2013; Wilson 2013; Jinek 2009). Therefore, the 19-23 -nucleotide length of small RNAs is the hallmark of mature miRNAs. This size definition was exploited to eliminate -18-nucleotide RNAs during sample preparation or analysis in most of the early next generation RNA sequencing (RNAseq) of miRNAs. On the other hand, RNAseq without RNA elimination found a substantial number of 10-18-nucleotide tiny RNAs (tyRNAs) bound to AGOs (Gangras 2017; Kuscu 2018; Baldrich 2019). Although some of tyRNAs are known to regulate gene expression similarly to mature miRNAs, little was known about whether tyRNAs play any specialized role.

Subsequently, in vitro studies revealed that 14-15 -nucleotide tyRNAs derived from specific miRNAs conferred a competitive slicing activity on AGO3. These tyRNAs are referred to as cleavage-inducing tyRNAs (cityRNAs). Furthermore, the RNAseq analyses showed that quite a few tyRNAs were bound to AGO3. It appears that many tyRNAs serve as cityRNAs. To date, many studies have focused on AGO2 based on the previous reports that only AGO2 can cleave RNAs (Wittrup 2015; Kannan 2018) and that the gene is essential (Cheloufi 2010). Since the mutation or deletion on the AGO2 gene is too fatal to cure with current treatment, infants with the mutation or deletion would not survive. Meanwhile, it has been reported that patients who suffer from neurological disease have mutations and deletions within the AGO3 gene (Tokita 2014), showing a possibility that AGO3 is not an essential gene for its survival but critical for normal body growth and neural development.

The data disclosed herein indicate that some cityRNAs function better with a booster to induce gene silencing. Eukaryotic cells use a chaperone system to load ~22-nt miRNA and siRNA duplexes into endogenous AGOs to form their RNA-induced silencing complex (RISC). However, there has been no system capable of loading cityRNAs into endogenous AGOs, although it was discovered the tinyRNA-biogenesis pathway (Sim et al., PNAS 2022). To overcome this issue, a system was developed. Boosters, whose structure mimics precursor miRNA, are RNA fragment(s) that protect certain cityRNA from the cellular nucleases and load it into endogenous AGOs properly so that the AGO and the cityRNA can be assembled into the functional RISC (cityRISC). Delooped Boosters were also developed, which showed competitive gene silencing. Both Boosters and Delooped Booster helped to transfect any specific cityRNA into any type of cells and form the cityRISC inside the cells.

Disclosed herein are “Booster” nucleic acids which help transfection of specific cityRNA into any types of cells and form cityRISC complex inside cells. The Booster nucleic acid can be any length that allows the cityRNA to complex with AGO. In some embodiments, the Booster comprises 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 or more nucleotides in length. In a preferred embodiment, the Booster comprises about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 nucleotides in length. Examples of Boosters can be seen in Figures 9 and 10 specifically. The booster can complex with a single-stranded cityRNA, which can provide the ability for the cityRNA to complex with AGO. The Booster can increase loading into the AGO complex by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any amount in between, below, or above these values. The Booster can have any sequence which is complementary to the cityRNA and prevents its degradation from nuclease.

The present disclosure provides gRNA compositions and complexes comprising a tinyRNA (tyRNA) and/or cleavage-induced tinyRNA (cityRNA) hybridized to a Booster nucleic.

It should be noted that for a cityRISC complex to form, the cityRNA must be separated from the Booster prior to loading into an AGO. In some embodiments, the cityRNA is 16 nucleotides or less in length. In some embodiments, the cityRNA is 14 nucleotides or less in length. In some embodiments, the cityRNA is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides in length. In some embodiments, the Booster nucleic acid hybridizes with the cityRNA. In some embodiments, a city RISC complex comprising a cityRNA and a Booster is referred to as a “cyBR” or a “cyDR”. In some embodiments, the Booster nucleic acid and cityRNA together form a secondary structure. As used herein, a “nucleic acid secondary structure” or a “secondary structure” refers to a structure formed from the base pairing interactions within a single nucleic acid or between two or more nucleic acids. Non-limiting examples of nucleic acid secondary structures include, but are not limited to a double helix, a stem loop, and pseudoknot. In some embodiments, the Booster nucleic acid and cityRNA together form a double helix. In some embodiments, the Booster nucleic acid and cityRNA together form a stem loop. In some embodiments, the Booster nucleic acid and cityRNA together for a pseudoknot. In some embodiments, the gRNA is greater than 18 nucleotides in length when Booster is hybridized to cityRNA. In some embodiments, the gRNA hybridized to the Booster comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the Booster nucleic acid is RNA or DNA. In some embodiments, the Booster nucleic acid comprises at least 2 nucleotides. In some embodiments, the Booster nucleic acid comprises 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, or more nucleotides. In some embodiments, the Booster nucleic acid comprises 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 nucleotides.

In some embodiments, the gRNA, including cityRNA and/or the Booster, can have at least one chemically modified nucleotide. These modified nucleotides may confer increased stability, decreased off-target effects, and/or reduced toxicity, as compared to a ssDNA or RNA not having the chemically modified nucleotide. They can also facilitate detection.

In some embodiments, the gRNA, including cityRNA and/or the Booster can comprise at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

In some embodiments, the chemically modified nucleobase is selected from 5- formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxycytidine (5moC), 5-hydroxycytidine (5hoC), 5-hydroxymethylcytidine (5hmC), 5-formyluridine (5fU), 5 -methyluridine (5-meU), 5- methoxyuridine (5moU), 5-carboxymethylesteruridine (5camU), pseudouridine ( ), Nl- methylpseudouridine (mel'P), N6-methyladenosine (me6A), or thienoguanosine (thG).

In some embodiments, the chemically modified ribose is selected from 2'-O-methyl (2'-O-

Me), 2'-Fluoro (2'-F), 2'-deoxy-2'-fluoro-beta-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'- SFANA, 2'-azido, UNA, 2 '-0-m ethoxy-ethyl (2'-0-ME), 2'-0-Allyl, 2'-0-Ethylamine, 2'-0- Cyanoethyl, Locked nucleic acid (LAN), Methylene-cLAN, N-MeO-amino BNA, or N-MeO- aminooxy BNA.

In some embodiments, the chemically modified phosphodiester linkage is selected from Phosphorothioate (PS), Boranophosphate, phosphodithioate (PS2), 3 ',5 '-amide, N3'- phosphoramidate (NP), Phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO).

RNA-induced Silencing Complexes (RISCs)

In the natural process of RNAi and gene silencing using RISC, long double-stranded RNAs are cleaved by the RNase III family member, Dicer, into nucleotides (nt) fragments with 5' phosphorylated ends and 2-nt unpaired and unphosphorylated 3' ends. AGOs then incorporate the guide strand into the RNA Interference Specificity Complex (RISC), while the passenger strand is released. The conditions which allow for loading of the double-stranded RNA molecule into RISC include the degradation of the passenger strand, thereby forming the cityRNA.

RISC uses the guide strand to find the target nucleic acid that has a complementary sequence leading to the endonucleolytic cleavage of the target mRNA. Therefore, the doublestranded RNA disclosed herein can be cleaved before exposure to RISC. Alternatively, only the cityRNA can be introduced to the RISC molecule.

The highly conserved AGO family members play a central role in the regulation of gene expression networks, orchestrating the establishment and the maintenance of cell identity throughout the entire life cycle, as well as in several human disorders, including cancers. Four functional AGOs (AG01, AG02, AG03, and AG04), with high structure similarity, have been described in humans and mice.

In some embodiments, the AGO, such as AG03 polypeptide used with the methods disclosed herein, is from a yeast. In some embodiments, the AGO polypeptide is from Vanderwaltozyma polyspora (also known as Kluyveromyces polysporus). Additional non-limiting examples of yeast AGO polypeptides can be from additional yeast species of the genus Kluyveromyces: K. aestuari, K. africanus, K. bacillisporus, K. blattae, K. dobzhanskii, K. hubeiensis, K. lactis, K. lodderae, K. marxianus, K. nonfermentans, K. piceae, K. sinensis, K. thermotolerans, K. waltii, K. wickerhamii, or K. yarrowii. Additional non-limiting examples of yeast AGO polypeptides can be from Yarrowia lipolytica, Pichia pastori, Candida vulgaris, Saccharomyces castellii, or Schizosaccharomyces pombe.

In some embodiments, the AGO polypeptide used with the methods disclosed herein is from a eukaryote. In some embodiments, the AGO polypeptide is from a mammal. In some embodiments, the AGO polypeptide is from a primate. In some embodiments, the AGO polypeptide is from a human.

In some embodiments, the AGO polypeptide is a full length AGO polypeptide. In some embodiments, the AGO polypeptide comprises a portion of the AGO protein. In some embodiments, the AGO polypeptide is a wild-type sequence. In some embodiments, the AGO polypeptide is a sequence with at least one mutation. In some embodiments, the AGO polypeptide comprises an amino acid sequence that is different from a naturally-occurring AGO polypeptide.

In some embodiments, the AGO polypeptide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of a full length AGO polypeptide. In some embodiments, the AGO polypeptide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of a wild-type AGO protein. In some embodiments, the full length AGO polypeptide comprises any one of the following protein accession identified numbers: Q9UL18, Q5TA57, Q6P4S0, Q9UKV8, Q8TCZ5, Q8WV58, Q96ID1, Q9H9G7, Bl ALIO, Q5TA55, Q9H1U6, Q9HCK5, A7MD27, or any derivatives thereof (including, but not limited to polypeptide derivatives originating from primates or other mammals).

In some embodiments, the RISC complex or any systems thereof may comprise additional complexes in addition to the AGO polypeptide. For example, additional components of the RISC complex may be present. Non-limiting examples of the additional components include, but are not limited to a Dicer protein, a ribosomal protein (such as, for example a 60S ribosomal protein, and 5S ribosomal protein), a helicase protein, a ribonucleoprotein, an RNA-binding protein, epigenetic regulatory proteins, transcription regulation proteins, and protein translation regulation proteins.

In one aspect, disclosed herein is a cell comprising the gRNA of any preceding aspect or the cityRISC complex of any preceding aspect. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a mammalian cell, a bacterial cell, or a yeast cell, including, but not limited to HEK cells, CHO cells, and HeLa cells. gRNA Kits, Compositions, and Components

Also disclosed herein is a kit comprising the gRNA of any preceding aspect. In some embodiments, the kit further comprises at least one Booster of any preceding aspect. In some embodiments, the gRNA comprises a tinyRNA (tyRNA) and/or cleavage-induced tinyRNA (cityRNA). In some embodiments, the gRNA of any preceding aspect comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides in length. In some embodiments, the kit further comprises a full length AGO peptide, a fragment or portion of an AGO peptide, or any derivative of an AGO peptide. In some embodiments, the kit further comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of a wild-type AGO peptide or a full length AGO peptide. In some embodiments, the kit can further comprise a complete RISC, a partial RISC, or any components thereof.

The kit can also include other components which can be used in the methods disclosed herein. For example, the kit can comprise components suitable for AGO and the double stranded nucleic acid to form a complex. In some embodiments, the kit further comprises reagents, buffers, and/or containers (including, but not limited to tubes and bags) suitable for forming a complex, suitable for storing one or more components/compositions or any preceding aspect, or suitable for executing a desired function (including, but not limited to regulating expression of a target nucleic acid and determining a suitable Booster nucleic acid).

For pharmaceutical applications, the present disclosure also provides a pharmaceutical composition comprising as an active agent having at least one gRNA of any preceding aspect, or a precursor thereof. The active agent may also comprise a DNA molecule encoding the gRNA molecule or the precursor thereof, and a pharmaceutical carrier. The composition may be used for diagnostic and therapeutic applications in human medicine or in veterinary medicine.

For diagnostic or therapeutic applications the composition may be in form of an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, a cream, its native form, or the like. The carrier may be any suitable pharmaceutical carrier. Preferably, a carrier is used of increasing the efficacy of RNA molecules to enter the target cells. Suitable examples of such carriers are liposomes, particularly cationic liposomes.

The composition of any preceding aspect may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the composition of any preceding will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease or disorder, the particular composition of any preceding, its mode of administration, its mode of activity, and the like. The composition of any preceding is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the composition of any preceding will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease or disorder being treated and the severity of the disease or disorder; the activity of the composition of any preceding aspect employed; the specific composition of any preceding aspect employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific composition of any preceding aspect employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition of any preceding aspect employed; and like factors well known in the medical arts.

The composition of any preceding aspect may be administered by any route. In some embodiments, the composition of any preceding aspect is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the composition of any preceding aspect (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.

The exact amount of composition of any preceding aspect required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

Methods of Use

In one aspect, disclosed herein is a method of regulating expression of a target nucleic acid using a cityRISC complex, wherein the cityRISC complex comprises an AGO and a gRNA, wherein said gRNA comprises a cityRNA hybridized with a Booster nucleic acid.

Also disclosed is a method of regulating expression of a target nucleic acid using an AGO molecule, wherein the AGO molecule has been loaded with a gRNA, the method comprising exposing the target nucleic acid to the AGO molecule loaded with the gRNA comprising a cityRNA optionally complexed with Booster, wherein the gRNA is complementary to a binding region of the target nucleic acid, and wherein the AGO molecule recognizes a non-binding region of the target nucleic acid.

In one aspect, disclosed herein is a method of determining a suitable Booster nucleic acid, the method comprising identifying a cityRNA, hybridizing said cityRNA to a Booster nucleic acid, detecting whether the cityRNA hybridized with the Booster nucleic acid is more efficient at binding with an AGO or regulating gene expression, and selecting the suitable Booster nucleic acid that binds with the AGO and regulating gene expression.

In some embodiments, the AGO peptide comprises AG01, AG02, AG03, or AG04. IN some embodiments, the AGO peptide comprises any one of the following protein accession identified numbers: Q9UL18, Q5TA57, Q6P4S0, Q9UKV8, Q8TCZ5, Q8WV58, Q96ID1, Q9H9G7, Bl ALIO, Q5TA55, Q9H1U6, Q9HCK5, A7MD27, or any derivatives thereof (including, but not limited to polypeptide derivatives originating from primates or other mammals). In some embodiments, the target nucleic acid is RNA or DNA.

In some embodiments, the method of any preceding aspect comprises the cityRNA comprising 16 nucleotides or less in length. In some embodiments, the method of any preceding aspect comprises the cityRNA comprising 14 nucleotides or less in length. In some embodiments, the cityRNA is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides in length. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid hybridizing with the cityRNA. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid and cityRNA together forming a secondary structure.

In some embodiments, the method of any preceding aspect comprises the gRNA having greater than 18 nucleotides in length when Booster is hybridized to cityRNA. In some embodiments, the method of any preceding aspect comprises the gRNA having 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length when the Booster is hybridized to the cityRNA. In some embodiments, the method of any preceding aspect comprises a Booster RNA or a Booster DNA. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid comprising at least 2 nucleotides. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid comprising 9 nucleotides.

In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid protecting the cityRNA from degradation by a nuclease. In some embodiments, the method of any preceding aspect comprises the Booster nucleic acid improving binding the cityRNA to the AGO. In some embodiments, the method of any preceding aspect improves binding the cityRNA to the AGO by at 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or more.

Once RISC has been loaded with the gRNA (such as city RNA used with Booster), it can be used for a variety of purposes. For example, it is known that gRNA can slice, or cleave, the target nucleic acid. This can effectively “silence” the target nucleic acid. This can be used to treat a variety of diseases and disorders. One can imagine that any time that a nucleic acid should be destroyed or silenced, the method disclosed herein can be employed. For example, dysfunctional gene expression can be modified in a disease and/or disorder including, but not limited to cancers (such as, for example acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva)), neurodegenerative diseases (such as, for example Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, spinal muscular atrophy, Alpers’ disease, Batten disease, Cerebro-oculo- facio-skeletal syndrome, Leigh syndrome, Prion diseases, monomelic amyotrophy, multiple system atrophy, striatonigral degeneration, motor neuron disease, multiple sclerosis (MS), Creutzfeldt-Jakob disease, Parkinsonism, spinocerebellar ataxia, dementia, and other related diseases), cardiovascular diseases (such as, for example coronary artery disease, high/low blood pressure, cardiac arrest/heart failure, congestive heart failure, congenital heart defects/diseases (including, but not limited to atrial septal defects, atrioventricular septal defects, coarctation of the aorta, double-outlet right ventricle, d-transposition of the great arteries, Ebstein anomaly, hypoplastic left heart syndrome, and interrupted aortic arch), arrhythmia, peripheral artery disease, stroke, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathies, hypertensive heart disease, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, eosinophilic myocarditis, valvular heart diseases, rheumatic heart diseases, and other related cardiovascular diseases), respiratory diseases (such as, for example asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pneumonia, bronchitis (chronic or acute bronchitis), emphysema, cystic fibrosis/bronchiectasis, pleural effusion, acute chest syndrome, acute respiratory distress syndrome, asbestosis, aspergilosis, severe acute respiratory syndrome (including, but not limited to SARS-CoV-1 and SARS-CoV-2), respiratory syncytial virus (RSV), middle eastern respiratory syndrome (MERS), mesothelioma, pneumothorax, pulmonary arterial hypertension, pulmonary hypertension, pulmonary embolism, sarcoidosis, sleep apnea, and other respiratory diseases), congenital diseases (such as, for example albinism, amniotic band syndrome, anencephaly, Angelman syndrome, Barth syndrome, chromosomal abnormalities (including, but not limited to abnormalities to chromosome 9, 10, 16, 18, 20, 21, 22, X chromosome, and Y chromosome), cleft lip/palate, club foot, congenital adrenal hyperplasia, congenital hyperinsulinism, congenital sucrase-isomaltase deficiency (CSID), cystic fibrosis, De Lange syndrome, fetal alcohol syndrome, first arch syndrome, gestational diabetes, Haemophilia, heterochromia, Jacobsen syndrome, Katz syndrome, Klinefelter syndrome, Kabuki syndrome, Kyphosis, Larsen syndrome, Laurence-Moon syndrome, macrocephaly, Marfan syndrome, microcephaly, Nager’s syndrome, neonatal jaundice, neurofibromatosis, Noonan syndrome, Pallister-Killian syndrome, Pierre Robin syndrome, Poland syndrome, Prader-Willi syndrome, Rett syndrome, sickle cell disease, Smith-Lemli-Optiz syndrome, spina bifida, congenital syphilis, teratoma, Treacher Collins syndrome, Turner syndrome, Umbilical hernia, Usher syndrome, Waardenburg syndrome, Werner syndrome, Wolf-Hirschhorn syndrome, Wolff-Parkinson-White syndrome, and other congenital diseases or disorders), gastrointestinal diseases (such as, for example heartburn, irritable bowel syndrome, lactose intolerance, gallstones, cholecystitis, cholangitis, anal fissure, hemorrhoids, proctitis, colon polyps, infective colitis, ulcerative colitis, ischemic colitis, Crohn’s disease, radiation colitis, celiac disease, diarrhea (chronic or acute), constipation (chronic or acute), diverticulosis, diverticulitis, acid reflux (gastroesophageal reflux (GER) or gastroesophageal reflux disease (GERD)), Hirschsprung disease, abdominal adhesions, achalasia, acute hepatic porphyria (AHP), anal fistulas, bowel incontinence, centrally mediated abdominal pain syndrome (CAPS), clostridioides difficile infection, cyclic vomiting syndrome (CVS), dyspepsia, eosinophilic gastroenteritis, globus, inflammatory bowel disease, malabsorption, scleroderma, volvulus, and other gastrointestinal diseases), and metabolic diseases (such as, for example diabetes mellitus Type I, diabetes mellitus Type II, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe syndrome, metachromatic leukodystrophy, Niemann-Pick syndrome, phenylketonuria (PKU), Tay-Sachs disease, Wilson’s disease, hemachromatosis, mitochondrial disorders or diseases (including, but not limited to Alpers Disease; Barth syndrome; beta. -oxidation defects:carnitine-acyl-carnitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency: Complex V deficiency; cytochrome c oxidase (COX) deficiency, LHON Leber Hereditary Optic Neuropathy; MM Mitochondrial Myopathy: LIMM Lethal Infantile Mitochondrial Myopathy; MMC Maternal Myopathy and Cardiomyopathy; NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease: FICP — Fatal Infantile Cardiomyopathy Plus, a MEL AS-associated cardiomyopathy: MELAS Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes; LDYT Leber's hereditary optic neuropathy and Dystonia; MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM Maternally inherited Hypertrophic CardioMyopathy; CPEO Chronic Progressive External Opthalmoplegia; KSS Kearns Sayre Syndrome; DM Diabetes Mellitus; DMDF Diabetes Mellitus+DeaFness; CIPO Chronic Intestinal Pseudoobstruction with myopathy and Opthalmoplegia; DEAF Maternally inherited DEAFness or aminoglycoside-induced DEAFness; PEM Progressive encephalopathy; SNHL SensoriNeural Hearing Loss; Encephalomyopathy; Mitochondrial cytopathy: Dilated Cardiomyopathy: GER Gastrointestinal Reflux: DEMCHO Dementia and Chorea; AMDF Ataxia, Myoclonus; Exercise Intolerance: ESOC Epilepsy, Strokes, Optic atrophy, & Cognitive decline; FBSN Familial Bilateral Striatal Necrosis: FSGS Focal Segmental Glomerulosclerosis: LIMM Lethal Infantile Mitochondrial Myopathy; MDM Myopathy and Diabetes Mellitus: MEPR Myoclonic Epilepsy and Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCM Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAIONNonarteritic Anterior Ischemic Optic Neuropathy; NIDDM Non-Insulin Dependent Diabetes Mellitus; PEM Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT Rett Syndrome: SIDS Sudden Infant Death Syndrome: MIDD Maternally Inherited Diabetes and Deafness; and MODY Maturity-Onset Diabetes of the Young, and MNGIE), and other metabolic diseases).

The target nucleic acid may further comprise a reporter gene, a pathogen-associated gene, e.g. a viral, protozoal or bacterial gene, or an endogenous gene, e.g. an endogenous mammalian, particularly human gene. The endogenous gene may be associated with a disorder, particularly with a hyperproliferative disorder, e.g. cancer, or with a metabolic disorder, e.g. a disorder associated with carbohydrate, energy, lipid, nucleotide, or amino acid metabolism or a disorder associated with the biosynthesis or metabolism of glycans, polyketides and non-ribosomal peptides, cofactors and vitamins or secondary metabolites, with the biodegradation of xenobiotics or with a neurodegenerative disorder such as Alzheimer, Parkinson, Huntington, ALS, MS etc. Thus, the present invention is suitable for the manufacture of reagents, diagnostics and therapeutics.

A further aspect of the invention relates to the modulating of a target gene specific silencing activity in a cell, an organism or a cell-free system, wherein the activity of at least one polypeptide of the gene silencing machinery is selectively modulated, e.g. increased and/or suppressed. By means of this selective activity increase and/or suppression, the efficacy of target nucleic acid specific silencing may be considerably increased. Thus, administration of double stranded molecules directed to the mRNA of a target gene, organism or a cell-free system (as indicated above) may be more effective.

The gene-specific silencing can comprise transcriptional gene silencing (TGS) activity or a post-transcriptional gene silencing (PTGS) activity. PTGS includes translational attenuation and/or RNA interference. Three phenotypically different but mechanistically similar forms of RNAi, co-suppression or PTGS in plants, quelling in fungi, and RNAi in the animal kingdom, have been described. The cityRNA can comprise a siRNA, shRNA or a miRNA molecule.

A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLES

The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Example 1: Gene Silencing by cityRNAs

AGOs use tyRNAs differently from miRNAs for target recognition

The homogeneous AGO3: 14-nt miR-20a complex was co-crystallized with a 16-nt target RNA whose tl-tl4 is fully complementary to the guide (Figure 1A). To avoid being cleaved during the crystallization, modifications were incorporated at tlO-tl l. The crystal structure showed a continuous electron density map of the gl-gl 1 of the guide and the tl-tlO of the target, but only the g2-g8 formed a duplex with the target RNA (Figure 1A and Figure 5A). Another isolated density map was seen in the central cleft, which corresponds to part of the unpaired gl2- gl4 (Figure 5 A). The co-crystallized target remained intact (Figure 5B), indicating that the current structure reflects a state before target cleavage. Meanwhile, no nucleotide density was found in the PAZ domain (Figure 5D). These observations indicate that the 3' end of 14-nt guide does not reach the PAZ domain and that the entire cityRNA lies in the central cleft, which is consistent with the previous study that the channel protects 13- 14-nt tyRNAs from being further trimmed by 3'— >5' exonucleases (Sim, G. et al. Manganese-dependent microRNA trimming by 3'— >5' exonucleases generates 14-nucleotide or shorter tiny RNAs. Proc Natl Acad Sci U S A 119, e2214335119, doi: 10.1073/pnas.2214335119 (2022)). Previous structural studies revealed that the AGO2-mature RISC widens the bilobed architecture in four steps as the guide extends the base pairing with no target (State I), the t2-t9 (State II), the t2-tl 8 and tl 3-tl 6 (State III), and the t2-t9 and t!2-t21 (State IV) (Figure 5D) (Schirle, N. T. & MacRae, 1. . The crystal structure of human AGO2. Science 336, 1037-1040, doi: 10.1126/science, 1221551 (2012); Schirle, N. T., Sheu- Gruttadauria, I. & MacRae, I. I. Structural basis for microRNA targeting. Science 346, 608-613, doi: 10.1126/science.1258040 (2014); Sheu-Gruttadauria, ., Xiao, Y., Gebert, L. F. & MacRae, I. I. Beyond the seed: structural basis for supplementary microRNA targeting by human AGO2. EMBO J 38, elOl 153, doi: 10.15252/embj.2018101153 (2019) (Hereinafter Sheu-Gruttadauria et al. 1); and Sheu-Gruttadauria, I. et al. Structural Basis for Target-Directed MicroRNA Degradation. Mol Cell 75, 1243-1255, doi: 10.1016/j.molcel.2019.06.019 (2019) (Hereinafter Sheu-Gruttadauria et al. 2)). The structure presented herein, whose guide is paired with the t2-t8, opens the two lobes similarly to State-Ill rather than State-II (Figure IB), showing that the central cleft of the current cityRISC structure is wide enough to accommodate the entire guide-target duplex. Again nevertheless, the g9-gl4 stays and is left flexible in the cleft without pairing with the target. These results demonstrate that tyRISCs do not create the standardized guide segmentation that ~22-nt siRNAs and miRNAs form in their mature RISCs whose PAZ domain captures the guide 3' end.

The nucleotides following the cityRNA-binding site work as a target-cleavage enhancer

It was previously revealed that 14-nt miR-20a and let-7a catalytically activated AGO3 but 14-nt miR-16 or miR-19b did not (Park, M. S., Sim, G., Kehling, A. C. & Nakanishi, K. Human AGO2 (AGO2) and AGO3 (AGO3) are catalytically activated by different lengths of gRNA. Proc Natl Acad Sci US H 117, 28576-28578, doi:10.1073/pnas.2015026117 (2020)(Hereinafter, Park et al.)). At that time, the non-homogeneous RISC was used, most of which had already been preoccupied with insect cell endogenous small RNAs during the overexpression. Since the RNA-free population of the purified AGO quite differed from batch to batch, the homogeneous RISCs were purified and were used for single-turnover kinetics in the present disclosure (Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Rapid and specific purification of AGO-small RNA complexes from crude cell lysates. RNA 19, 271-279, doi: 10.1261/rna.036921.112 (2013)). AGO3 cleaved about 60-nt complementary targets when loaded with 14-nt miR-20a or let-7a but not when loaded with 14-nt miR-16 or miR-19b (Figure 6A), which is consistent with the work from Park et al. using the non-homogeneous RISCs. However, the AGO3: 14-nt miR-20a barely cleaved a 14-nt target RNA (Figures 2A and 2B), explaining why the g9-gl4 was not paired with the target in the crystal structure (Figure 1A). Notably, the initial velocity of the AGO3: 14-nt miR-20a increased up to 27.5-fold as the 5' end was extended to be 16-, 18-, 20-, and 23-nt in the target length (Figure 2B). This result shows that the tl 5-t23 contributes to target cleavage, although this extended target region has no base-pairing partner on 14-nt miR-20a (Figure 2A). On the other hand, the AGO3: 14-nt let-7a cleaved a 14-nt complementary target 10-fold faster than the AGO3: 14-nt miR-20a, but the target extension from 14 to 21 nt increased the initial velocity only 2-fold (Figures 2C and 2D). In contrast, neither of a 14- nor 23-nt complementary target was cleaved by AGO3 loaded with 14-nt miR-19b or miR-16 (Figures 6B, 6C, 6D, 6E, and 6F). Therefore, the target cleavage by AGO3 primarily depends on the cityRNA sequence and can be enhanced in the presence of tl 5-t23 (Figure 61).

The crystal structure disclosed herein showed no density of the tl l-t!4 (Figure 1A and Figure 5A), indicating the dynamic movement of this segment when the target length was 14 nt. Meanwhile, the extended tl5-t23 enhanced the target cleavage, though it had no base-pairing partner (Figures 2A, 2B, 2C, and 2D). Therefore, it was contemplated that AGO3 directly interacts with the tl5-t23 and reduces the dynamic of the target strand so that the tl l-tl4 can form the extensive base pairing, enhancing the target cleavage (Figure 6G). To test the idea, chimeric targets were made using the 23-nt miR-20a target as a template. The tl 5-t23 was replaced with the tl 5-t23 of the 23-nt miR-19b target (20a-19b target), the tl 5-t21 of the 21-nt let-7a target (20a- 7a target), or the tl 5-t22 of the 22-nt miR-16 target (20a-16 target) (Figure 2E). These chimeric targets were cleaved at different efficiencies (Figure 2F). Especially, the AGO3: 14-nt miR-20a cleaved the 20a-20a target ~8 times faster (yO = 4.60 ± 0.22 pM-s'¹) than the 20a-7a target (yO = 0.59 ± 0.02 pM-s'¹). A similar trend was observed when chimeric targets were made based on the let-7a target, albeit the target cleavage was not enhanced as much as that of the miR-20a-based chimeric ones (Figures 2G and 2H). These results indicate that specific sequences of tl5-t23 enhance the AGO3 slicer activity and show that AGO3 recognizes the sequences of the tl 5-t23 rather than just interacting with the sugar-phosphate backbone of this target segment. In contrast, the AGO3 loaded withl4-nt miR-19b and miR-16 cleaved their cognate chimeric targets at very low efficiencies or at non-detectable level, respectively (Figures 6H, 61, 6J, 6K, and 6L). These results further support that the cityRNA sequence is the primary determinant of the target cleavage by AGO3. Since the enhancement of target cleavage depends on the tl 5-t23 sequence following the cityRNA-binding site, the region “Target-cleavage Enhancer in Cis (TEC)” was named (Figure 21). The presence of 5' and 3' flanking regions further enhanced the targe cleavage by AGO3: 14- nt miR-20a (Figure 2B), whereas this is not the case for AGO3”14-nt let-7a (Figure 2D). Given that both 58- and 60-nt targets of miR-20a and let-7a have the 5' and 3' flanking regions whose sequences are the same, the tl 5-t23 of the miR-20a is indispensable for the flanking regions to enhance the initial velocity further. A TEC also would help AGO3-cityRISC recognize the flanking regions on the positively charged surface to facilitate the target cleavage.

Some cityRNA catalytically activated AGO2 better than their miRNAs

It was previously reported that, unlike AGO3, AGO2 resulted in a lower slicer activity with cityRNAs than with their parental miRNAs (Park et al). Similarly, the homogeneous AGO2 cleaved the 58-nt complementary target with 21-nt let-7a (vO = 240.28 ± 7.88 pM-s'¹) better than with 14-nt let-7a (yO = 37.48 ± 2.58 pM-s'¹) (Figure 7A). On the other hand, AGO2 cleaved the 60-nt complementary target with 14-nt miR-20a (yO = 71.80 ± 4.70 pM-s'¹) slightly better than with 23-nt miR-20a (yO = 53.79 ± 0.82 pM-s'¹) (Figure 7B). These results show that some tyRNAs work as cityRNAs for AGO2.

To investigate how target lengths affect the slicer activity of AGO2, different lengths of targets were tested for cleavage by AGO2-mature RISC and -cityRISC, as did for AGO3. The

AGO2:23-nt miR-20a increased the initial velocity 7.2-fold when the target is extended from 20 to 23 nt (Figures 3A and 3B). Meanwhile, the AGO2:21-nt let-7a raised the initial velocity 7.3- fold when the target is extended from 18 to 20 nt (Figures 3C and 3D). These results indicate that 20-23-nt lengths of targets can be important for sufficient slicing activity by releasing the 3' end of mature miRNAs from the PAZ domain, as seen in the previous structures of AGO2-mature RISCs (Sheu-Gruttadauria et al. 1 and Sheu-Gruttadauria et al. 2).

The AGO2: 14-nt miR-20a increased the initial velocity little by little as the target is extended from 14 to 23 nt (Figures 3E and 3F). In contrast, the initial velocity of the AGO2: 14-nt let-7a became 2 times faster when the target length was extended from 14 to 16 nt but remained the same even for 16-58-nt targets (Figures 3G and 3H). This difference in the enhancement of target cleavage between the two AGO2-cityRISCs was reminiscent of that seen in AGO3- cityRNAs (Figures 3 A, 3B, 3C, and 3D) and prompted the thinking that the fully complementary binding site of 23-nt miR-20 has a TEC also for AGO2-cityRNA. When the abovementioned two sets of the chimeric targets were tested for cleavage, AGO2-cityRISCs showed the same trends (Figures 31, 3 J, 3K, and 3L), as seen in AGO3 (Figures 2E, 2F, 2G, and 2H), showing that TEC is applicable to both AGO2- and AGO3-cityRISCs. Swapping the tl 5-t23 also changed the initial velocities of the AGO2-mature RISCs, but the region enhanced the target cleavage differently between the two AGO2-mature RISCs.

Boosters help cityRNA-dependent silencing

To evaluate the significance of cityRNAs for gene silencing, 14-nt single-stranded modified let-7a (14-nt let-7a^m: Figures 8A and 8B) were co-transfected into HEK293T cells with a dual-luciferase reporter (DLR) plasmid whose Renilla luciferase gene has the 7a-7a target site in its 3'UTR (Figure 5 A). Modifications were incorporated into the guide to avoid degradation in the cells ¹’⁶. As expected, 21 -nt let-7a duplex reduced the luciferase activity (Figure 5B). But neither 14-nt let-7a^m nor 14-nt let-7a^m duplex was competent for silencing (Figure 5B). In Park et al., however, the immunoprecipitated FLAG-AGO3 from HEK293T cells, in which 14-nt modified miR-20a (miR-20a^m) was co-transfected with a FLAG-AGO3 plasmid, cleaved a complementary target RNA in vitro (Park et al.). It was considered that although cityRNAs themselves could not be loaded into AGO efficiently or properly enough to cause silencing, a small population of the 14-nt miR-20a^m-loaded FLAG-AGO3 in the immunoprecipitant must be sufficient to cleave the 5'-end radiolabeled target in vitro (Park et al.). To overcome this possible loading issue, 14-nt let-7a^m was annealed with another RNA called Booster, and formed a pseudo hairpin mimicking precursor miRNA, which was named “cityRNA-Booster for RNAi” (cyBR) (Figures 8A and 8B). Transfection of a cyBR carrying 14-nt let-7a^m (cyBR-7a^m) repressed the relative luciferase activity down to ~ 50%, whereas Booster alone failed to cause silencing (Figure 5B). These results demonstrate that the observed silencing was attributed to the cityRNA and that Booster helped to load it into endogenous AGOs.

The in vitro kinetics data showed that when programmed with 14-nt let-7a, both AGO2 and AGO3 cleaved the 7a-20a target more efficiently than the 7a-7a one (Figures 2H and 3L). It was tested whether 14-nt let-7a showed a similar trend in gene silencing. Transfection of cyBR- 7a^m repressed the relative luciferase activity by -75% when the 7a-7a target site was switched to the 7a-20a one (Figure 5C, Lane 6 of Figure 5B, and Lane 8 of Figure 5D), which is consistent with the in vitro target cleavage. Neither of 14-nt let-7a^m, 14-nt let-7a, nor their duplexes showed silencing (Figure 5D). Notably, cyBR-7a^m reduced the relative luciferase activity by -90% in HCT116 wild-type, A549, and HeLa cells (Figure 8C). However, cyBR-7a^m did not show significant silencing when the 20a-20a target site resides in the 3'UTR in HEK293T and HCT116 wild-type (Figure 8D and 8E), proving that the cityRNA-dependent silencing is sequencedependent. A similar silencing level was observed when 14-nt let-7a^m was replaced with 14-nt unmodified let-7a (14-nt let-7a) to form a cyBR (cyBR-7a) (Lane 9 of Figure 5DThese results indicate that the modifications on cityRNAs are not necessarily required for silencing activity, presumably because the Booster would protect the docked unmodified cityRNAs from degradation.

Also, a Delooped-Booster was made, whose structure is the same as Booster, except that it has a 3 ' 2-nt overhang, instead of the tetranucleotide loop, and it was named cityRNA-Delooped- Booster for RNAi (cyDR) (Figure 8A). A cyDR including 14-nt let-7a^m or 14-nt let-7a (cyDR- 7a^m and cyDR-7a, respectively) showed similar silencing activities in HEK293T cells (Figure 5E), as did cyBR-7a^m and cyBR-7a, but a delooped-Booster alone did not (Figure 5D). The competence of cyDR-7a^m and cyDR-7a in silencing was also confirmed in HCT116 wild-type, A549, and HeLa cells (Figure 8F). Altogether, both cyBR and cyDR help cityRNAs to induce gene silencing in DLR system to similar extents.

AGO3-cityRISC cause silencing

It has now been shown that cityRNAs are capable of gene silencing (Figures 5B, 5D, and 5E). Since human cells express four AGOs, the observed silencing in the DLR assays was a composite competency of all the paralogs. To evaluate the pure impact of AGO3 -city RISC on gene silencing, a DLR assay was performed using the 7a-20a reporter gene in the HCT116 AGO 1/2 (-/-) cells, which is known to express AGO4 protein at an undetectable level (Chu, Y. et al. AGO binding within 3 '-untranslated regions poorly predicts gene repression. Nucleic Acids Res 48, 7439-7453, doi: 10.1093/nar/gkaa478 (2020) and Liu, Z., Johnson, S. T., Zhang, Z. & Corey, D. R. Expression of TNRC6 (GW182) Proteins Is Not Necessary for Gene Silencing by Fully Complementary RNA Duplexes. Nucleic Acid Ther 29, 323-334, doi: 10.1089/nat.2019.0815 (2019)). cyBR-7a^m showed -60% reduction in the luciferase activity, whereas 21 -nt let-7a duplex lowered the activity by -40% (Figure 5F). This is consistent with the in vitro cleavage where AGO3 cleaved the 7a-20a target with 14-nt let-7a 10 times faster than with 21-nt let-7a (Figure 2H and 6N). A similar impact of cyDR-7a^m on silencing was seen in the double-knockout cells, which was slightly better than cyDR-7a (Figure 5G). Transfected cyBR-7a^m, however, failed to repress the relative luciferase activity when the reporter gene had a 20a-20a target site (Figure 8E). These results demonstrate that AGO3-cityRISC is capable of silencing.

The impact of cityRNAs on silencing differs among the cell types

Specific 3'— >5' exonucleases trim AGO-associated miRNAs, generating their tyRNAs, some of which work as cityRNA to catalytically activate AGO3 (Sim, G. K., A. C.; Park, M. S.; Secor, J.; Divoky, C.; Zhang, H.; Malhotra, N.; Bhagdikar, D.; Abd El-Wahaband, E.; Nakanishi, K. Manganese-dependent microRNA trimming by 3 ' — 5' exonucleases generates 14-nucleotide or shorter tiny RNAs. bioRxiv (2022)). Therefore, guide trimming seems to remodel the RNAi of the cell. It was tested whether 21-nt let-7a changes the targets to be silenced when the length becomes 14 nt in HCT116 AG01/2(-/-). HCT116 wild-type, and HEK293T cells. In each of the three cells, 21-nt let-7a duplex silenced both 7a-7a and 7a-20a reports at similar efficiencies of -40% (Figures 5H, 51, and 5J). Meanwhile, cyBR-7a^m showed quite different silencing percentages depending on the cell type (Figures 5H, 51, and 5J). Especially, in HCT116 wild-type cells, cyBR-7a^m repressed -90% of the 7a-20a reporter expression while silencing only 30% of the 7a-7a reporter one (Figure 51). On the other hand, such a huge difference in silencing the two target sites by cyBR-7a^m was seen in nether HCT116 AG01/2(-/-) nor HET293T cells (Figure 5H and 5J). Although the possibility that cyBR and cyDR can load cityRNAs into AGOs more efficiently than the endogenous chaperone system required for sufficient loading of miRNA and siRNA duplexes cannot be excluded (Iwasaki, S. et al. Defining fundamental steps in the assembly of the Drosophila RNAi enzyme complex. Nature 521, 533-536, doi: 10.1038/naturel4254 (2015)), this data shows that guide trimming changes the target RNAs to be silenced in HCT116 wild-type cells. Methods

In vitro cleavage assay to determine cityRNA sequence

The target RNAs were radiolabeled using y-³²P ATP (3,000 Ci mmol'¹; PerkinElmer) with T4 Polynucleotide kinase (NEB) at 37 °C for 1 h, followed by 90 °C for 1 min to inactivate the kinase. Unincorporated y-³²P ATP was removed using MicroSpin™ G-25 columns (Cytiva). For the RISC assembly, 1 pM AGO3 WT was incubated with 10 nM gRNA in IX Reaction Buffer (25 mM HEPES-KOH pH 7.5, 100 mM KC1, 5 mM MgCh, 5 mM DTT, 0.005% (v/v) NP-40, 0.01 mg/ml baker’s yeast tRNA, 0.05 mg/mL BSA, 0.5 U/pL Ribolock), with a total volume of 40 pL, for 1 hr at 37°C. For the target cleavage, 10 nM radiolabeled target was added to the RISC assembly mixture and incubated 30 and 60 min at 37 °C. The reaction was quenched at each time point with the same volume of 2x urea quenching dye (8 M urea, 1 mM EDTA, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromophenol blue, 10% (v/v) phenol) including 20% glycerol, heated at 90 °C for 2 min, and resolved on an 8 M urea, 20% (29: 1) acrylamide/bis-acrylamide denaturing gel. Gels were dried, exposed to a storage phosphor screen, and imaged on a Typhoon Imaging System (GE Healthcare). The raw image file was used to quantify the substrate and product bands, and Image Lab (Bio-Rad) was used for background correction. All data were graphed using GraphPad Prism.

In vitro cleavage assay to determine cleavage-inducing t!5-t23 sequence

The target RNAs were radiolabeled using y-³²P ATP (3,000 Ci mmol'¹; PerkinElmer) with T4 Polynucleotide kinase (NEB) at 37 °C for 1 h, followed by inactivation of the kinase at 90 °C for 1 min. Unincorporated y-³²P ATP was removed using MicroSpin™ G-25 columns (Cytiva). 2.5 nM radiolabeled target was added to 10 nM homogeneous AGO3/14-nt miR-20a RISC in IX Reaction Buffer, with a total volume of 40 pL, at 37 °C for the target cleavage. The reaction was quenched with the same volume of 2x urea quenching dye (8 M urea, 1 mM EDTA, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromophenol blue, 10% (v/v) phenol) at each time point, heated at 90 °C for 2 min, and resolved on an 8 M urea, 20% (29: 1) acrylamide/bis-acrylamide denaturing gel. Gels were dried, exposed to a storage phosphor screen, and imaged on a Typhoon Imaging System (GE Healthcare). The raw image file was used to quantify the substrate and product bands, and Image Lab (Bio-Rad) was used for background correction. All data were graphed using GraphPad Prism. Cell culture

HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS (Gibco). Cell lines were incubated at 37 °C and 5% CO2.

Dual luciferase assay

HEK293T cells were seeded on 24-well plate with 500 pL of medium and grown up to about 70% confluency. The old media was replaced with fresh supplemented DMEM. The cells were co-transfected with 50 ng of psiCHECK-2 encoding the target sequence and 6 pmol RNA using 2.5 pL of TransIT-X2 (Minis) and 100 pL of Opti-MEM™ (Gibco). 24 hrs posttransfection, cells were washed with 500 pL of IX phosphate buffered saline (PBS) per well, followed by cell lysis using 200 pL of Passive Lysis Buffer (PLB) per well. Luciferase activities were measured using GloMax® Navigator System (Promega). All luciferase emission measurements were performed using the Dual -Luciferase® Reporter assay (Promega). 10 pL of cell lysate were transferred to LLMITRAC™ (Greiner Bio-One) 96-well plates for luminescence recordings. 100 pL of Luciferase Assay Reagent II reagent (Promega) was added to each well to measure Flue activity. Then, the same volume of Stop & Gio® reagent (Promega) was added to measure Rluc activity. Rluc luminescence was divided by Flue luminescence, followed by normalizing to the cells transfected with only psiCHECK-2 encoding the target sequence.

Homogeneous RISC purification

Human AGO2 and AGO3 were expressed in Tni cells using a Baculovirus Expression System (Thermo Fisher Scientific). T. ni cells from 2-6 L cultures were resuspended in Harvest Buffer (Buffer A with 1 mM PMSF and SigmaFAST Protease Inhibitor Cocktail, EDTA-free (Sigma)) after harvest. The cells were then lysed in a C3 Homogenizer at 4 °C, followed by centrifugation at 23,000 rpm. The supernatant was added to 10-20 mL Ni Sepharose HP beads (Cytiva) pre-equilibrated in Harvest Buffer. The mixture of beads and supernatant was placed on a shaker to incubate at 4°C and 100 rpm for 1 h. The beads were then recovered by centrifugation at 4,000 xg for 3 min. Beads were washed 4 times with Buffer A (50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 0.5 mM TCEP) and 2 times with Buffer B(50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 25 mM imidazole, 0.5 mM TCEP). Resuspended beads in Buffer B with 5 mM CaCh and added 10 pL micrococcus nuclease (Takara) per Liter of original cell culture. Performed digestion at RT for 1 h, followed by 6 washes of 4 CV Buffer B. The beads were then loaded onto a gravity column and AGO protein was eluted with Buffer C (50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 300 mM imidazole, 0.5 mM TCEP). The amount of eluted AGO protein was estimated by SDS-PAGE using known concentrations of BSA. GRNA was added to samples containing AGO protein at a 1 :2 RNA: AGO ratio and incubated on ice for 15 min. Sample was dialyzed O/N at 4°C in the presence of TEV to cleave the His-tag. The programmed AGO protein was subjected to a 2 x 5 mL HisTrap HP (Cytiva) and 2 x 1 mL HiTrap Q FF (Cytiva) columns to remove the cleaved His-tag and excess gRNA, respectively. The homogeneous AGO protein was then purified using the ARPON method, with slight modifications. Elution oligo was removed by running the RISC sample through a third HiTrap Q FF column. Flow-through was concentrated and loaded to a Superdex 200 increase 10/300 GL column (Cytiva). RISC concentrations were measured using A280. Each RISC was directly frozen in liquid nitrogen or diluted to 2 pM in Cryo-EM buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP) containing 1 mg/mL BSA and 50% glycerol before being frozen in liquid nitrogen. The RISCs were stored at -80 °C.

Crystallization of AGO3:14-nt miR-20a:16-nt target

1 mg/mL AGO3: 14-nt miR-20a: 16-nt target complex (1: 1.2 ratio of RISC Target) was incubated on ice for 3-5 min before setting up sitting-drop crystallization plates with 1 : 1 sample:reservior solution. Crystals showed up O/N in Condition A8 of ICSG Core I Suite (Qiagen). The condition was then optimized, to 150 mM tri-sodium citrate and 12% (w/v) PEG 3350. Crystals were harvested in buffer containing 150 mM tri-sodium citrate and 40% (w/v) PEG 3350 and flash frozen in liquid nitrogen. Crystal diffractions were collected at the Advanced Photon Source (Argonne National Laboratory).

Single-turnover kinetics

2.5 nM radiolabeled target RNA was incubated in IX Reaction Buffer with 10 nM RISC in 40 pL reactions at 37 °C. 5 pL aliquots were quenched with 2x quenching dye at 0.5, 1, 2, 3, 5, 10, and 20 min. Cleavage products were resolved on 8 M urea, 20% (29: 1) acrylamide/bis- acrylamide denaturing gels. Phosphor images were taken by the Typhoon Imager (GE Healthcare) and band intensity was quantified using Image Lab (Bio-Rad).

Equilibrium binding assay

The binding of RISC to target RNAs was determined using filter binding assays as previously described (Schirle et al., 2014) with modification. Specifically, 0.1 nM ³²P labeled target was incubated with increasing concentrations of RISC in IX Binding Buffer (25 mM HEPES-KOH pH 7.5, 100 mM KC1, 5 mM DTT, 0.005% (v/v) NP-40, 0.01 mg/ml baker’s yeast tRNA) at RT for 60 min in a total volume of 50 pL. The samples were then loaded onto a dot blot apparatus (GE Healthcare) under vacuum with the Protran nitrocellulose membrane (0.45 mm pore size, Whatman, GE Healthcare Life Sciences) on top and Hybond Nylon membrane (Amersham, GE Healthcare) at the bottom. The top nitrocellulose membrane captures RISC-target complex, while the bottom nylon membrane captures the unbound target RNA. The membranes were then washed 10 times with 75 pL ice-cold Wash Buffer (25 mM HEPES-KOH (pH 7.5), 100 mM KC1, 5 mM DTT, 0.01 mg/ml baker’s yeast tRNA). The membranes were then air-dried, and phosphor images taken by the Typhoon Imager (GE Healthcare). Signals were quantified using Image Lab (Bio-Rad).

Data Analysis

When [E] « [S], time courses were fit to y = mx + b; when [S] < [E], time courses were fit to y = yo + where the initial rate, vO = Ak [Lu, W.P., and Fei, L. (2003). A logarithmic approximation to initial rates of enzyme reactions. Anal. Biochem.]. The initial rates of target cleavage at different target concentrations were fit to Michaelis-Menten model with Prism version 9.5.0 (GraphPad Software, Inc.):

where [Nr] is total target concentration, V_max is the calculated maximum velocity, K_m is the Michaelis-Menten constant.

Dissociation constants were calculated using the following equation (Wee et al., 2012 and lessica Sheu-gruttadauria et al. 2019) with Prism version 9.5.0 (GraphPad Software, Inc.) :

where F is fraction of target bound, Bmax is maximum number of binding sites, [Er] is total enzyme concentration, [Sr] is total target concentration, and ED is the apparent equilibrium dissociation constant.

Example 2: Dual-luciferase Reporter Assay by Transfection of cityRNAs with Booster

A pseudo hairpin comprising a 14-nt modified let-7a and a Booster reduced the relative luciferase (Luc) activity (Figure 12). At that time, the HEK293T cells were incubated at room temperature (RT) before transfection, to avoid disassembly of the pseudo hairpin RNA. This time, however, the same experiment was repeated with and without RT incubation to see whether the pseudo hairpin RNA can cause gene silencing even at 37 °C.

Protocol with RT (Room Temperature) incubation: Day 1

Cell culture

1. Discard the media (DMEM, 10% FBS, no antibiotics) from 100% grown HEK293T cells

(Pl > P2, 10 cm plate)

2. Trypsinize with 1 mL trypsin and incubate at 37 degrees for 1 min

3. Detach the cells by tapping the plate

4. Add 9 mL DMEM (10% FBS, no antibiotics) and resuspend the cells

5. Add 100 uL of cells to 500 uL of DMEM (10% FBS, no antibiotics) in 24-well plate

6. Incubate in the 37 degrees incubator for O/N (1 :50 pm)

14-nt mod-1 let-7a + let-7a booster annealing

1. Mix 50 pmol of 14-nt mod-1 let-7a and 50.5 pmol of let-7a booster in IX annealing buffer

(10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM DTT, 10 mM MgC12) with total 50 uL volume

- Final 1 uM

2. Incubate at 90 degrees for 5 min and cool down on ice

3. Leave the duplex at RT until the transfection

Plasmid preparation

1. Take the plasmid out from the freezer and leave at RT until the transfection

Day 2

Pre-warm up

1. Warm up the DMEM (10% FBS, no antibiotics) in the 37 degrees dry bath for 1.5 hr (9:00 - 10:30 am)

- The media is almost 500 mL. After 1.5 hr, it was about RT

2. Take OMEM and TransIT-X2 out from the fridge and leave at RT for 1.5 hr (9:00 - 10:30 am)

3. Take the 24-well plate out from the 37 degrees incubator and leave at RT in a hood for 0.5 - 1.5 hr

- About 70 % confluency Sample preparation

1. In the lab, add 100 ng of psiCHECK-2 plasmid (50 ng/well) and 12 pmol of 14-nt mod-1 let-7a + let-7a booster (6 pmol/well) in a 1.5 mL tube

2. Bring the tube to the cell culture room

Transfection

After 1.5 hr, replace the media from the 24-well plate with 10 mL of fresh media (DMEM, 10% FBS, no antibiotics) (10:30 am)

2. Leave the plate at RT in a hood until the transfection

3. Add 200 uL (100 uL/well) of OMEM to the tube with plasmid and duplex

4. Add 5 uL (2.5 uL/well) of TransIT-X2 to the tube and mix well

5. Incubate for 15-20 min at RT in a hood (10:50 - 11 :05 am)

6. Dropwise 103.3 uL of the mixture to each well

7. Leave the plate at RT in a hood for 10 min (11 :05 - 11 : 15 am)

8. Incubate in the 37 degrees incubator for 48 hrs and harvest

Protocol without RT incubation:

Day 1

Cell culture

(Pl > P2, 10 cm plate)

2. Trypsinize with 1 mL trypsin and incubate at 37 degrees for 1 min

3. Detach the cells by tapping the plate

4. Add 9 mL DMEM (10% FBS, no antibiotics) and resuspend the cells

6. Incubate in the 37 degrees incubator for O/N (1 :50 pm)

14-nt mod-1 let-7a + let-7a booster annealing

1. Mix 50 pmol of 14-nt mod-1 let-7a and 50.5 pmol of let-7a booster in IX annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM DTT, 10 mM MgC12) with total 50 uL volume

- Final 1 uM

2. Incubate at 90 degrees for 5 min and cool down on ice

3. Leave the duplex at RT until the transfection

Plasmid preparation

1. Take the plasmid out from the freezer and leave at RT until the transfection

Day 2

Pre-warm up

- The media is almost 500 mL. After 1.5 hr, it was about RT

- About 70 % confluency

Sample preparation

2. Bring the tube to the cell culture room

Transfection

1. After 1.5 hr, replace the media from the 24-well plate with 10 mL of fresh media

(DMEM, 10% FBS, no antibiotics) (10:30 am)

2. Put the plate back to the 37 degrees incubator until the transfection

3. Add 200 uL (100 uL/well) of OMEM to the tube with plasmid and duplex

4. Add 5 uL (2.5 uL/well) of TransIT-X2 to the tube and mix well

5. Incubate for 15-20 min at RT in a hood (10:50 - 11 :05 am)

6. Dropwise 103.3 uL of the mixture to each well 7. Directly put the plate back to the 37 degrees incubator

8. Incubate in the 37 degrees incubator for 48 hrs. and harvest

Results:

There were two replicates for each condition. In all tested conditions, the HEK293T cells were co-transfected with psiCHECK-7a-20a, whose 3' untranslated region (3'UTR) includes fully complementary sequences to the gl-gl4 of let-7a and the gl5-g23 of miR-20a (Figure 11). The transfection of 21-nt let-7a duplex reduced the relative luciferase activity (Lane 2 of Figure 11), indicating that the DLR assay worked properly (i.e., positive control). When 14-nt single-stranded (ss) modified let-7a was transfected, no gene silencing was observed (Lane 2 of Figure 11). However, the relative luciferase activity was reduced to 45% when the pseudo hairpin RNA was transfected (Lane 4 of Figure 11). These results were consistent with those of the last experiment also performed with RT incubation (Lanes 5-8 of Figure 12), demonstrating the significance of Booster for gene silencing was reproduced by 14-nt let-7a.

* In HEK293T cells, AG02 is abundantly expressed.

The same experiment was repeated but without RT incubation (Lanes 5-8 of Figure 11). The results were similar to those without RT incubation (Lanes 1-4 of Figure 11). Notably, the transfection of the pseudo hairpin reduced the relative Luc activity less than 40% (Lane 8 of Figure 11), indicating that Booster can enhance the loading of 14-nt let-7a into AGOs. This result shows the possible application of this technique to therapeutic (i.e., a pseudo hairpin delivered to the patient body at 37 °C can retain the structure).

The DLR assay was previously performed using a pseudo hairpin composed of 14-nt single-stranded modified miR-20a (14-nt ss mod miR-20a). A weak gene silencing up to 10-20% reduction in the relative Luc activity was observed. These results show that 14-nt miR-20a is capable of gene silencing, but the pseudo hairpin might be disassembled due to the low Tm of 14- nt miR-20a. Using the website (www.oligoevaluator.com/LoginServlet), the annealing temperatures (Tms) of the following 14-nt tiny RNAs were calculated.

14 nt miR-20a : 33.7 °C

14 nt let-7a : 46.2 °C

14 nt miR-16 : 45.3 °C

14 nt miR-19b : 43.5 °C The Tm of 14-nt miR-20a is significantly lower than the others. LNAs were incorporated in the complementary sequence in Booster to increase the Tm, while the 14-nt miR-20a remains the same modification (but not LNA).

Example 3: tinyRNAs activate AGO’s autonomous target recognition to control cleavage for silencing

TinyRNAs (tyRNAs) are <17-nucleotide (nt) gRNAs associated with AGOs, yet their functional significance has remained enigmatic. Certain 14-nt cleavage-inducing tyRNAs (cityRNAs) catalytically activate human AG03. Here, the crystal structure of AG03 in complex with 14-nt miR-20a and its complementary target were presented, revealing a distinct target RNA recognition from microRNA-loaded counterparts. CityRNA-loaded AG02 and AG03 check target complementarity with guide nt 2-8 while directly recognizing target sequences immediately upstream of the tyRNA-binding region, subsequently rendering the target paired with guide nt 9- 14, then cleaved. Systems were developed to load endogenous AGOs with desired tyRNAs and to demonstrate that unlike microRNAs, cityRNA-mediated silencing heavily relies on target cleavage. These results uncovered AGO’s intrinsic capability to autonomously recognize target sequences to manipulate cleavage for gene silencing.

MicroRNAs (miRNAs), small interfering RNA (siRNAs), and piwi-interacting RNAs (piRNAs) exemplify small non-coding RNAs that regulate gene expression, with lengths ranging from 20 to 30 nt (7-3). While piRNAs follow a unique biogenesis pathway (E. F. Pettersen et cd., UCSF Chimera— a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612 (2004)), miRNAs and siRNAs share a common biogenesis machinery. Specifically, their precursors are processed by Dicer into ~22-nt miRNA- and siRNA-duplexes, which are then loaded into AGOs with chaperone assistance (S. Iwasaki et al., Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol Cell 39, 292-299 (2010) and K. Nakanishi, Anatomy of RISC: how do small RNAs and chaperones activate AGO? Wiley Interdiscip Rev RNA 7, 637-660 (2016)). Following passenger-strand ejection, the remaining miRNA and siRNA form the mature RNA-induced silencing complex (mature RISC), having both 5' and 3' ends recognized at the MID and PAZ domains, respectively (Sheu- Gruttadauria et al. 1). Utilizing the guide nucleotide positions 2-8 (g2-g8) known as the seed region, the mature RISC surveys the complementary sequences of target RNAs (D. P. Bartel, Metazoan MicroRNAs. Cell 173, 20-51 (2018) and R. Shang, S. Lee, G. Senavirathne, E. C. Lai, microRNAs in action: biogenesis, function and regulation. Nat Rev Genet, (2023)). Among four human AGOs, AGO3 shares the same catalytic tetrad as AGO2 but has shown limited slicing activity with ~22-nt gRNAs. Therefore, AG02 was thought to be the only slicer (J. Liu et al., AG02 is the catalytic engine of mammalian RNAi. Science 305, 1437-1441 (2004) and G. Meister et al. , Human AG02 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15, 185- 197 (2004)).

Meanwhile, previous studies in humans and plants have identified AGO-associated tyRNAs with lengths shorter than standard small non-coding RNAs (P. Baldrich et cd., Plant Extracellular Vesicles Contain Diverse Small RNA Species and Are Enriched in 10- to 17- Nucleotide "Tiny" RNAs. Plant Cell 31, 315-324 (2019); Z. Li et aL, Characterization of viral and human RNAs smaller than canonical MicroRNAs. J Virol 83, 12751-12758 (2009); and K. Nakanishi, Are AGO-Associated Tiny RNAs Junk, Inferior miRNAs, or a New Type of Functional RNAs? Front Mol Biosci 8, 795356 (2021)). Defined as 17 nt or shorter, tyRNAs lack the length to position their 3' end at the PAZ domain (G. Sim et aL, Determining the defining lengths between mature microRNAs/small interfering RNAs and tiny RNAs. Sci Rep Accepted, (2023)). RNA sequencing analyses indicate that tyRNAs are derived from endogenous miRNAs and tRNAs, and even viral miRNAs (Z. Li et aL, Characterization of viral and human RNAs smaller than canonical MicroRNAs. J Virol 83, 12751-12758 (2009); C. Kuscu et aL, tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicerindependent manner. RNA 24, 1093-1105 (2018); and Y. Han et al., tRF3008A suppresses the progression and metastasis of colorectal cancer by destabilizing FOXK1 in an AGO-dependent manner. J Exp Clin Cancer Res 41, 32 (2022)). A tyRNA biogenesis pathway was recently identified in which specific 3'— >5' exonucleases trim AGO-associated miRNAs to 13-14-nt tyRNAs (G. K. Sim, A. C.; Park, M. S.; Secor, J.; Divoky, C.; Zhang, H.; Malhotra, N.; Bhagdikar, D.; Abd El-Wahaband, E.; Nakanishi, K., Manganese-dependent microRNA trimming by 3 ' — 5' exonucleases generates 14-nucleotide or shorter tiny RNAs. bioRxiv, (2022)). tyRNAs were initially thought to regulate gene expression similarly to miRNAs, but some act as cleavageinducing tyRNAs (city RNAs), enhancing AGO3 endonuclease activity up to ~82-fold (M. S. Park, G. Sim, A. C. Kehling, K. Nakanishi, Human AGO2 and AGO3 are catalytically activated by different lengths of gRNA. Proc Natl Acad Sci USA 117, 28576-28578 (2020)).

The present disclosure reports that cityRISCs exhibit distinct target RNA recognition and silence unique targets, compared to mature RISCs. cityRISCs show enhanced target cleavage influenced by the sequence immediately upstream of the tyRNA-binding site, a contrast to mature RISCs, which use the extensive base pairing for cleavage facilitation (L. M. Wee, C. F. Flores- Jasso, W. E. Salomon, P. D. Zamore, AGO divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151, 1055-1067 (2012)). Lastly, two auxiliary RNA systems were introduced that facilitate the loading of desired tyRNAs into endogenous AGOs and show that the resultant cityRISCs prefer unique targets to silence mainly by target cleavage. These findings have revealed another layer of gene regulation by cityRNAs.

Structure of target-bound cityRISC

The homogeneous AGO3 : 14-nt miR-20a complex was purified and it was determined that the 3.45 A co-crystal structure with a target RNA whose nucleotide positions 1-14 (tl-tl4) are fully complementary to the gl-gl4 of the guide while having two adenylates at its 3' end (Figure 1 A, Figure 19A, and Table 1). To avoid target cleavage during crystallization, modifications were incorporated at target nucleotide positions 10 and 11 (tlO-tl l) (Figures 19B, 19C, and Table 2). The crystal structure showed a continuous electron density map of the gl-gl l of the guide and the tl-tlO of the target, but only the g2-g8 formed a duplex with the target RNA (Figure 1 A and Figure 19D). Another isolated density map was seen in the central cleft, which corresponds to part of the unpaired gl 3 or gl4 (Figure 19D). The co-crystallized target remained intact (Figure 19E), indicating that the current structure reflects a state before target cleavage. Previous studies revealed that AGO2-mature RISC creates a standardized guide segmentation and uses the seed (g2-g8), 3' supplementary (gl3-gl6), tail (gl 7-3 ' end), and central (g9-gl2) regions, in this order, to recognize a complementary target site and cleave it (Figure 19F) (D. P. Bartel, Metazoan MicroRNAs. Cell 173, 20-51 (2018)). Structural studies revealed that AGO2-mature RISC widens the bilobed architecture in four steps as the guide extends the base pairing from a state with no target pairing (State I), to pairing of the t2-t9 (State II), then the t2-t8 and tl 3-tl 6 (State III), and finally the t2-t9 and tl2-t21 (State IV) (Figures 19F and 19G) (N. T. Schirle, I. J. MacRae, The crystal structure of human AGO2. Science 336, 1037-1040 (2012); N. T. Schirle, J. Sheu- Gruttadauria, I. J. MacRae, Structural basis for microRNA targeting. Science 346, 608-613 (2014); J. Sheu-Gruttadauria, Y. Xiao, L. F. Gebert, I. J. MacRae, Beyond the seed: structural basis for supplementary microRNA targeting by human AGO2. EMBO J 38, elOl 153 (2019); and J. Sheu- Gruttadauria et cd.. Structural Basis for Target-Directed MicroRNA Degradation. Mol Cell 75, 1243-1255 (2019)). Ultimately, the AGO2-mature RISC cleaves the target between tlO and ti l when the central region pairs with the target. The current structure, whose guide is paired with only the t2-t8 but not the rest, widens the two lobes similarly to State III rather than State II (Figure IB). The observed structural differences between the mature RISC and cityRISC indicate that the loaded ~22-nt guide has the MID and PAZ domains located within a certain distance to retain a closed RISC conformation. On the other hand, the 14-nt tyRNA is not long enough to locate its 3' end at the PAZ domain (Figure 1A) (G. Sim et cd.. Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs. Sci Rep Accepted, (2023)), leaving the bilobed structure open. As a result, the central cleft of cityRISC is wide enough to accommodate the entire guide-target duplex. These structural observations indicate that cityRISCs have a unique target recognition different from mature RISCs.

Target cleavage by AGO3-cityRISC

Park et al. discovered that 14-nt miR-20a and let-7a, as opposed to miR-16 or miR-19b, activated AGO3 catalytically (M. S. Park, G. Sim, A. C. Kehling, K. Nakanishi, Human AGO2 and AGO3 are catalytically activated by different lengths of gRNA. Proc Natl Acad Sci U S A 117, 28576-28578 (2020)). At that time, non-homogeneous RISCs were utilized, most of which were already occupied by endogenous small RNAs during overexpression in insect cells (Park et al. Proc Natl Acad Sci USA 117, 28576-28578 (2020)). In the present disclosure, homogeneous RISCs were used to analyze the single-turnover cleavage rate (vo), and a consistent trend was observed in which 14-nt miR-20a and let-7a act as cityRNAs, while 14-nt miR-19b and miR-16 function as non-cityRNAs (Figure 15A and 15B).

Since the crystal structure revealed no base pairs beyond position g8 (Figure 1 A), it was explored whether the unmodified 14-nt target could be cleaved by AGO3: 14-nt miR-20a. The AGO3 -cityRISC showed cleavage of the 14-nt target RNA, but the cleavage efficiency was much lower than that of the 60-nt one (Figure 15B and 15D). To delve into this cleavage discrepancy, the 14-nt target was extended on its 5' end to 16, 18, 20, and 23 nt, fully complementary to the gl- gl6, gl-gl8, gl-g20, and gl-g23 of 23 -nt miR-20a, respectively (Figure 15C). Surprisingly, an increased extension correlated with higher vo, and the vo of the 23 -nt target cleavage by AGO3 : 14- nt miR-20a increased up to ~24-fold compared to that of the 14-nt target (Figure 15D). These results show that the presence of the 5' upstream flanking region significantly contributes to target cleavage, even though this extended region cannot base-pair with the 14-nt guide (Figure 15C). In comparison, although AGO3 : 14-nt let-7a cleaved its 14-nt complementary target 10-fold faster than AGO3 : 14-nt miR-20a, extending the target from 14 to 21 nt only increased the initial velocity 2-fold (Figures 15E and 15F). Conversely, AGO3 loaded with either 14-nt miR-19b or miR-16 did not show decent cleavage of their extended targets (Figures 15 A, 15B, 20B, and 20E). This lack of target cleavage did not seem to stem from weak target binding because AGO3 : 14-nt miR- 16 binds to its target at a similar level to AGO3 : 14-nt miR-20a binding to its corresponding target (Figure 20G). These results indicate that target cleavage by AGO3-cityRISC primarily hinges on the city RNA sequence and can be augmented by the presence of the 5' unpaired target region. Target cleavage by AGO2-cityRISC

AG03 -city RISC enhanced miR-20a target cleavage in the presence of the 5' upstream region of tyRNA-binding site. The same assays were performed to assess the impact of the 5' upstream region on target cleavage by AGO2-cityRISC. AGO2: 14-nt miR-20a cleaved the 14-nt target faster than AG03: 14-nt miR-20a (Figure 15D and 15G). The single turnover cleavage rate increased up to 14.3-fold as the target extended from 14 to 23 nt (Figure 15G). In contrast, the cleavage rate of AG02: 14-nt let-7a showed a 2-fold increase when the target extended from 14 to 16 nt but remained unchanged for 16-21 -nt targets (Figure 15H). Thus, the two AGO2-cityRISCs exhibited distinct cleavage rates, mirroring the pattern observed in their AGO3-cityRISC counterparts (Figures 15D and 15F). While AG02: 14-nt miR-19b and AG02: 14-nt miR-16 increased their cleavage rates 2-3-fold upon target extension (vo = 2.33 ± 0.05 pM-s'¹ and 0.62 ± 0.08 pM-s'¹, respectively) (Figures 20C and 20F), these rates were lower than that of AGO2: 14- nt miR-20a against the 14-nt target (vo = 2.67 ± 0.22 pM-s'¹). This result further supports that the cityRNA sequence is the primary determinant for target cleavage by cityRISCs.

Next, it was examined how AGO2-mature RISC cleaves targets of different lengths. AGO2:23-nt miR-20a increased the initial velocity by 7.2-fold when the target extended from 20 to 23 nt (Figures 20H and 201). Meanwhile, AGO2:21-nt let-7a raised the initial velocity by 7.3- fold when the target extended from 18 to 20 nt (Figure 20 and 20K). These results are consistent with the previous reports that AGO2-mature RISC remains inactive unless the g20-g23 pairs with targets to release the 3' end from the PAZ domain (I. Sheu-Gruttadauria, Y. Xiao, L. F. Gebert, I. I. MacRae, Beyond the seed: structural basis for supplementary microRNA targeting by human AGO2. EMBO J 38, elOl 153 (2019) and I. Sheu-Gruttadauria et al., Structural Basis for Target- Directed MicroRNA Degradation. Mol Cell 75, 1243-1255 (2019)), while diverging from AGO2- cityRISC target cleavage patterns (Figures 15G and 15H).

Some AGO2-cityRISCs are superior slicers to their mature RISCs

In using non-homogeneous RISCs, AGO2 exhibited lower slicer activity with cityRNAs than their parental miRNAs, unlike AGO3 (Park et al.). Similarly, homogeneous AGO2:21-nt let- 7a cleaved the 58-nt target much faster (vo = 240.28 ± 7.88 pM-s'¹) than AGO2: 14-nt let-7a (vo = 37.48 ± 2.58 pM-s-1) (Figure 21 A). Interestingly, AGO2: 14-nt miR-20a showed slightly better cleavage of the 60-nt target (vo = 71.80 ± 4.70 pM-s'¹) than AGO2:23-nt miR-20a (vo = 53.79 ± 0.82 pM-s'¹) (Figure 2 IB), showing that particular cityRNAs enhance the slicing efficiency of AGO2 over their parental miRNAs. The sequence upstream of tyRNA-binding site determines the extent of target cleavage

Enhanced target cleavage by city RISC was evident in the 23 -nt miR-20a target but not in the 21 -nt let-7a target (Figure 15). Considering their distinct sequences upstream of the tyRNA- binding site, it was contemplated that the sequence in this region determines the extent of cleavage enhancement and named this region tyRNA-binding site adjacent motif (TAM). To test this idea, chimeric targets sharing a tyRNA-Binding site (tl-tl4) complementary to the gl-gl4 of miR-20a (termed 20a^B) were designed. Superscript B stands for tyRNA-Binding site) (Figure 16A). Three chimeric targets have a different TAM sequence, each complementary to the gl5-g22 of 22-nt miR-16 (20a^B-16^T), the gl5-g23 of 23-nt miR-19b (20a^B-19b^T), or the gl5-g21 of 21-nt let-7a (20a^B-7a^T) (Superscript T stands for TAM). AG03 : 14-nt miR-20a efficiently cleaved these targets in the order 20a^B-20a^T, 20a^B-16^T, 20a^B-19b^T, and 20a^B-7a^T, with an ~8-fold difference in cleavage rates (Fig. 16B). Similar trends were observed when replacing their 20a^B with a 14-nt let-7a- Binding site (7a^B) (Figs. 16D-E) or 14-nt miR- 19b-Binding site (19b^B) (Figures 22A and 22B), though the enhancement was smaller than 20a^B-based chimeric targets. AGO2-cityRISCs showed the same trends in cleavage enhancement for these chimeric targets (Figures 16C, 16F, and 22C). However, 14-nt miR-16-associated AG02 and AG03 showed very modest and no cleavage, respectively, of the chimeric targets with a 14-nt miR-16-Binding site (16^B), irrespective of the TAM sequence (Figures 22D and 22F), confirming the primary influence of the gl-gl4 sequence on the target cleavage by tyRNA-associated RISCs.

AG02-mature RISCs showed different efficiencies in the cleavage of these chimeric targets compared to their cityRISC counterparts (Figures 22G, 22H, 221, and 22 ). Since mature RISCs use a guide long enough to pair with TAM, the impact of the TAM sequence on target cleavage depends on its complementarity with the gl5-g23 (Figure 22K). In contrast, cityRISCs, unable to engage their cityRNA in TAM recognition, demand specific sequences of the unpaired TAM to enhance their target cleavage drastically (Figure 22L). cityRISCs and mature RISCs prefer different target sites for cleavage

The rate for the single-turnover cleavage of the 7a^B-7a^T target by AGO2:21-nt let-7a (vo = 148.51 pM-s'¹) was 20-fold higher than that of the 7a^B-20a^T target (vo = 7.81 pM-s'¹) (Figure 22 ). However, the rate for cleavage of the 7a^B-20a^T target by AG02: 14-nt let-7a (vo = 88.61 pM-s'¹) was more than doubled compared to the 7a^B-7a^T target (vo = 41.70 pM-s'¹) (Figure 16F). These findings show that AGO2, depending on guide length, changes the preferred target cleavage site (Figure 17A). The TAM serves as a cleavage enhancer when targeted by cityRISCs, but it pairs with part of the 3' supplementary (gl 3-gl 6) and tail (gl 7-3 ' end) regions when targeted by mature RISCs. To understand how the TAM plays different roles, Uns and K_ms was measured for the cleavage of the 7a^B-7a^T and 7a^B-20a^T targets by AGO2:21-nt let-7a or AG02: 14-nt let-7a (Figure 23 and Table 3). Calculating the relative kcat ( a^B-2Qa^B/'7a^B-'7a^B') and the relative K_m (7a^B-20a^T/7a^B- 7a^T), it was found that switching the target site from the 7a^B-7a^T to the 7a^B-20a^T retained the K_m but significantly lowered the at for AG02-mature RISC (Figure 17B). The similarity between the relative vo and feat implies that pairing the TAM with the 3' supplementary and tail regions primarily influences the catalytic step rather than the cleavage-product release. On the other hand, AGO2-cityRISC increased the relative feat to ~2, similar to the relative vo, while maintaining the relative K_m at ~1 (Figure 17C). The same trend was observed in target cleavage by AG03- cityRISC (Figure 17D). These results show that the TAM predominantly contributes as a cleavage enhancer to the catalytic step in target cleavage by cityRISC.

To examine whether the cityRNA sequence influences the impact of the TAM on target cleavage, the tyRNA-binding site was changed from 7a^B to 20a^B. AGO2-cityRISC increased both relative vo and feat from ~2 to ~8, while maintaining their relative K_m (Figures 17C and 17E). This indicates that the effect of TAM on target cleavage varies based on the cityRNA sequence. Likewise, AG03 loaded with 14-nt miR-20a showed comparable relative vd, kcat, and K_m when the 20a^B-7a^T target was switched to the 20a^B-20a^T (Figure 17F). In both AG02- and AG03- cityRISCs, TAM’ s significant contribution to their kcat enhancement was observed when the 20a^B- 7a^T target site was changed to 20a^B-20a^T. These findings reinforce the notion that even the same tyRNA-binding sites are cleaved by a cityRISC at different efficiencies depending on their TAM sequence (Figure 17A) - a distinctive characteristic compared to their mature RISC counterparts, which cleave targets based on guide-target base pairing.

Target-strand dynamics promotes the cleavage by cityRISC

The 20a^T facilitates a catalytic reaction step in target cleavage by cityRISCs more efficiently than the 7a^T. However, the 14-nt cityRNA is not long enough to pair with the TAM, which prompted the thinking that when the TAM has a specific sequence, such as 20a^T, the AGO in a cityRISC directly recognizes the sequence to enhance the target cleavage. To validate this, filter-binding assays were performed, quantifying dissociation constants (Kd) of AGO3-cityRISC for different targets (Table 4) (L. M. Wee, C. F. Flores-Iasso, W. E. Salomon, P. D. Zamore, Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151, 1055-1067 (2012)). AGO3: 14-nt let-7a displayed an affinity for the 14-nt 7a^B target Kd = 355.3 ± 45.6 pM), which increased 9-fold by the target extension with 7a^T Kd = 40.4 ± 13.5 pM). Unexpectedly, extending the 14-nt 7a^B target with 20a^T only increased the affinity by 3-fold (Kd = 114.8 ± 28.1 pM). Similarly, the affinity of AG03 : 14-nt miR-20a for the 14-nt 20a^B target Kd = 187.1 ± 45.0 pM) was increased 9-fold by the target extension with 7a^T Kd = 26.2 ± 5.7 pM) but remained similar to that of the extension, 20a^T Kd = 165.5 ± 36.3 pM). These results show that AGO3 in cityRISC interacts tightly with the 7a^T but weakly or negligibly with the 20a^T. Given that the existence of 20a^T enhanced the turnover number (Figures 17C, 17D, 17E, and 17F), the following model is proposed: when the AGO of cityRISC barely recognizes the TAM, the resultant dynamics of the target strand renders the downstream t9-t 14 quickly paired with the cityRNA g9-gl4. As a result, the TAM works as a strong cleavage enhancer (Figure 17G, top). Conversely, when the AGO of cityRISC recognizes the TAM, the downstream t9-tl4 becomes less accessible to the g9-gl4. Thus, the TAM moderately enhances the catalytic reaction (Figure 17G, bottom).

Boosters help endogenous AGOs to load tyRNAs

To assess the relevance of cityRNAs for in vivo gene silencing, a dual -luciferase reporter (DLR) plasmid containing the 7a^B-7a^T target site in the 3' untranslated region (3'UTR) of the Renilla luciferase gene was co-transfected along with 14-nt single-stranded modified let-7a (14- nt let-7a^m)(Superscript m stands for chemically modified RNA) into HEK293T cells (Figures 24A, 24B, 24C, and Table 2). Modifications were introduced into the guide to prevent any possible degradation in the cells (Park et al. and G. Sim etal., Manganese-dependent microRNA trimming by 3'— >5' exonucleases generates 14-nucleotide or shorter tiny RNAs. Proc Natl Acad Sci USA 119, e2214335119 (2022)). 14-nt let-7a^m transfected as a single-stranded RNA or a duplex did not trigger gene silencing, but transfection of the 21 -nt let-7a duplex (positive control) significantly reduced relative luciferase activity, as expected (Figure 18A). Since 14-nt guides are not long enough to have both termini recognized by the MID and PAZ domains (Figure 1A) (G. Sim etal., Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs. Sci Rep Accepted, (2023)), it was thought that tyRNAs alone could not be loaded into AGOs efficiently. Then, an RNA Booster was devised that, upon annealing with 14-nt let-7a^m, forms a pseudo-hairpin resembling a precursor miRNA (Figure 18A and Figures 24A and 24B). The pseudo-hairpin was termed “cityRNA-Booster for RNAi (cyBR).” The Booster alone did not induce silencing (Figure 18A), but transfection of a cyBR carrying 14-nt let-7a^m (cyBR-7a^m) successfully loaded the cityRNA into endogenous AGO2 (Figure 24D) and reduced relative luciferase activity lower than 50% in HEK293T cells (Figure 18A). Notably, cyBR-7a^m also elicited significant gene silencing in HCT116, HeLa, and A549 cells (Figure 25A). cyBR-20a^m and -92a^m, housing modified cityRNAs from miR-20a and miR-92a (Park et al.), respectively, induced silencing in HCT116, HeLa, and A549 cells but not in HEK293T cells (Figures 25B and 25C), showing cell type-dependent gene silencing. Silencing by cyBR-7a^m was minimal for the reporter gene with a 20a^B-20a^T target site (Figure 25D), indicating that cityRNA- dependent silencing operates in a sequence-dependent manner. In contrast, non-cityRNA-derived cyBR-16^m and -19b^m resulted in weak or negligible silencing activity in all tested cells (Figures 25E and 25F). The disparities in silencing efficacy between cityRNAs and non-cityRNAs show the significant reliance of cityRNA-driven silencing on target cleavage.

The in vitro kinetic data demonstrated that when programmed with 14-nt let-7a, both AG02 and AG03 cleaved the 7a^B-20a^T target more efficiently than the 7a^B-7a^T (Figures 16E and 16F). This observation prompted an investigation of whether the TAM influences cityRNA-driven gene silencing (Figure 24C). The 14-nt let-7a^m, 14-nt let-7a, their duplexes, or Booster alone did not cause silencing, if any (Figure 18B). However, transfection of cyBR-7a^m increased silencing from -50% to -75% when the TAM was switched from 7a^T to 20a^T (Figure 18B), consistent with the in vitro target cleavage assays (Figures 16E and 16F). Notably, cyBR-7a^m reduced luciferase activity of the 7a^B-20a^T reporter by up to 90% in HCT116, A549, and HeLa cells (Figure 18C). A similar silencing level was observed when 14-nt let-7a^m was replaced with 14-nt unmodified let- 7a (14-nt let-7a) to form a cyBR (cyBR-7a) (Figure 18B), indicating that modifications on cityRNAs are not required for silencing, presumably because the Booster protected the docked, unmodified cityRNAs from degradation. This was verified by in-cell RISC assembly assays showing that 14-nt let-7a was loaded into endogenous AGO2 only when transfected as cyBR-7a (Figure 24D).

Additionally, a De-looped-Booster was designed by replacing the tetranucleotide loop of Booster with a 3' 2-nt overhang, forming cityRNA-De-looped-Booster for RNAi (cyDR) (Figure 18D and Figures 24A and 24B). Both cyDRs, including 14-nt let-7a^m (cyDR-7a^m) or 14-nt let-7a (cyDR-7a), showed similar silencing activities in HEK293T cells (Figure 18D), as did cyBR-7a^m and -7a (Figure 18B), while the De-looped-Booster alone did not. Guide loading into endogenous AGO2 by cyDR-7a^m and cyDR-7a was confirmed by in-cell RISC assembly assay (Figure 24D). The competence of cyDR-7a^m and -7a in silencing was also observed in other cells (Figure 18E). As seen from cyBR-7a^m (Figures 18A and 18B), switching the TAM from 92a^T to 20a^T drastically enhanced silencing by cyDR-92a^m (Figure 26), further supporting the significance of the TAM sequence for gene silencing.

To determine the cellular localization of cityRISCs, a plasmid encoding FLAG-AGO2 or

-AGO3 was co-transfected with a cyDR-7a, whose 14-nt let-7a had a Cyanine3 (Cy3) at its 3' end, into HeLa cells (Figure 27). The FLAG-AGOs loaded with the 14-nt let-7a-Cy3 were detected mainly in the cytoplasm (Figures 18F and 18G). Altogether, these data illustrate that both cyBR and cyDR help cityRNAs to induce gene silencing in the cytoplasm. cityRNAs and miRNAs target different sites for silencing

The in vitro assays demonstrated that 21 -nt let-7a-loaded AG02 preferred to cleave the 7a^B-7a^T target over the 7a^B-20a^T (Figure 17A). However, in cellular assays across four tested cell lines, 21 -nt let-7a silenced both fully and partially complementary targets at similar levels (Figure 18H). In contrast, cyBR-7a^m exhibited preferred silencing of the 7a^B-20a^T reporter gene to the 7a^B-7a^T (Figure 18H), showing that the RISCs switch silencing targets when 21 -nt let-7a is shortened to its 14-nt cityRNA. This switch of silencing targets was more obvious between 22-nt miR-92a and its 14-nt cityRNA (Figure 181). The same silencing switch was observed when cyDR-92a^m was transfected, instead of cyBR-92a^m (Figure 18 J). These findings indicate that shortening miRNAs to tyRNAs alters the preferred target site for gene silencing.

In the in vitro target cleavage assays, AGO2-cityRNA, AG03 -cityRNA, and AG02- mature RISC failed to cleave the 7a^B-7a^T and 7a^B-20a^T targets when mismatches were introduced at tlO and tl 1 (Figure 28 and Table 2). This result indicates that these RISCs cannot silence the reporter gene through a slicer-dependent mechanism when the target carries two nucleotide mismatches at tlO and tl 1. However, incorporating the same mismatches into the 7a^B-7a^T and 7a^B- 20a^T target sites on the reporter gene had only a slight or negligible impact on silencing by the 21- nt let-7a duplex across four different cell lines (Figure 18K), showing that 21 -nt let-7a represses gene expression through slicer-independent pathways, compensating for the lack of target cleavage. In contrast, cyBR- and cyDR-7a^m exhibited a noticeable reduction in silencing activity for the mismatched 7a^B-7a^T target (Figure 18K). The reduction became more significant when the TAM changed from 7a^T to 20a^T (Figure 18L). These results strongly support that cityRNA-driven silencing predominantly relies on target cleavage.

Discussion

In the present disclosure, it was demonstrated that AGOs utilize not only guide-dependent but also guide-independent target recognition. This challenges the prevailing consensus that AGOs recognize target sequences solely through gRNA (A. Grishok etal., Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23-34 (2001) and S. M. Hammond, S. Boettcher, A. A. Caudy, R. Kobayashi, G. J. Hannon, AGO2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146-1150 (2001)). The findings also indicate an intrinsic capability of AG02 and AG03 to autonomously recognize specific nucleotide sequences. This inherent ability remains concealed when AGO-associated gRNA maintains the standard miRNA length of ~22 nt, as the 3' half of the guide interacts with the TAM, reducing its accessibility to the AGO (Fig. 22K). In contrast, loading an AGO with a 14-nt tyRNA results in exclusive recognition of the TAM by its recognition site on AGO, as the tyRNA cannot pair with the TAM (Fig. 17G and Fig. 22L). Consequently, mature RISCs and their tyRISCs take different approaches to target recognition. This adaptability in miRNAs and AGOs fundamentally influences target specificity for silencing. miRNAs elicit gene silencing through three distinct downstream mechanisms: target cleavage, translational repression, and mRNA destabilization (D. P. Bartel, Metazoan MicroRNAs. Cell 173, 20-51 (2018) and K. Nakanishi, Anatomy of four human AGO proteins. Nucleic Acids Res 50, 6618-6638 (2022)). In animals, mRNA destabilization predominantly contributes to gene repression (S. Jonas, E. Izaurralde, Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16, 421-433 (2015)), as mature RISCs rarely cleave target RNAs due to the limited sequence complementarity between miRNAs and mRNAs (K. Nakanishi, When Argonaute takes out the ribonuclease sword. J Biol Che nr 105499 (2023)). Only a limited number of targets have been identified to be cleaved by mature RISCs (F. V. Karginov et al., Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases. Mol Cell 38, 781-788 (2010); S. M. Hammond, E. Bernstein, D. Beach, G. J. Hannon, An RNA-directed nuclease mediates post- transcriptional gene silencing in Drosophila cells. Nature 404, 293-296 (2000); and S. Yekta, I. H. Shih, D. P. Bartel, MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594-596 (2004)). Despite 14-nt tyRNAs having approximately 65,000 times more chances of finding fully complementary sequences compared to ~22-nt miRNAs, the in vitro assays revealed that cityRNAs, not non-cityRNAs, induce significant gene silencing mainly through target cleavage (Fig. 25 and Figs. 18K-L). Moreover, the study unveiled that AGO2 and AGO3 autonomously recognize specific sequences immediately upstream of the tyRNA-binding site, thereby attenuating target cleavage (Fig. 16). These distinctive features in cityRISCs prevent cleaving fully complementary tyRNA-binding sites when specific sequences are present upstream. Since stress and viral infection induce specific 3'— >5' exonucleases that convert mature RISCs to tyRISCs (G. K. Sim, A. C.; Park, M. S.; Secor, J.; Divoky, C.; Zhang, H.; Malhotra, N.; Bhagdikar, D.; Abd El-Wahaband, E.; Nakanishi, K., Manganese-dependent microRNA trimming by 3 ' — 5' exonucleases generates 14-nucleotide or shorter tiny RNAs. bioRxiv, (2022); A. M. Stankiewicz, J. Goscik, A. Majewska, A. H. Swiergiel, G. R. Juszczak, The Effect of Acute and Chronic Social Stress on the Hippocampal Transcriptome in Mice. PLoS One 10, e0142195 (2015); and S. Deymier, C. Louvat, F. Fiorini, A. Cimarelli, ISG20: an enigmatic antiviral RNase targeting multiple viruses. FEBS Open Bio 12, 1096-1111 (2022)), AGO2 and AGO3 may have evolved to learn and remember specific sequences, preventing cleavage of these targets during emergencies. A safety system to avoid cleaving specific targets is reminiscent of the mechanism by which CRISPR (Clustered regularly interspaced short palindromic repeats)-CRISPR-associated (Cas) systems distinguish foreign DNAs from the host genomic one (B. Wiedenheft, S. H. Sternberg, J. A. Doudna, RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331-338 (2012)). Although Cas9 recognizes DNA targets complementary to a short CRISPR RNA sequence, reading a specific protospacer adjacent motif (PAM) is a prerequisite for unwinding the DNA duplex at the target site (M. Jinek et al.. A programmable dual -RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012) and H. Nishimasu et al., Crystal structure of Cas9 in complex with gRNA and target DNA. Cell 156, 935-949 (2014)). Like DNA targets that lack a PAM and thus escape from cleavage by Cas9, RNA targets with specific sequences may avoid being cleaved by cityRISCs.

The present disclosure also introduces two Booster systems, cyBR and cyDR, allowing the programming of endogenous AGOs with specific tyRNAs. This advancement enables the exploration of the physiological roles of tyRNAs. Employing those two systems in DLR assays, it was discovered that cityRNAs induce not only gene silencing but also alter silencing targets compared to mature RISCs. This data indicate that the predominant mechanism of cityRNA- mediated silencing involves target RNA cleavage, distinguishing it from miRNAs. Thus, the findings unveil novel gene silencing mechanisms mediated by cityRNAs.

Materials and Methods

Homogeneous RISC purification: Homogeneous RISCs were purified by ARPON method (Flores-Jasso 2 13). Human AGO2 and AGO3 were expressed in T. ni cells using a Baculovirus Expression System (Thermo Fisher Scientific). T. ni cells from 2-6 L cultures were resuspended in Buffer A (50 mM Tris-HCl pH 8.0, 0.3 MNaCl, 0.5 mM TCEP). 1 mMPMSF and SigmaFAST Protease Inhibitor Cocktail, EDTA-free (Sigma) were added to the cells after harvest. Cells were lysed in a C3 Homogenizer at 4 °C, followed by centrifugation at 23,000 rpm. The supernatant was added to 10-20 mL Ni Sepharose HP beads (Cytiva) pre-equilibrated in Buffer A. The mixture of beads and supernatant was incubated on a shaker at 4 °C and 100 rpm for 1 hour. The beads were then recovered by centrifugation at 4,000 * g for 3 min. Beads were washed 4 times with Buffer A and twice with Buffer B (50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 25 mM imidazole, 0.5 mM TCEP). The beads were resuspended in Buffer B with 5 mM CaCh, after which 10 pL micrococcus nuclease (Takara) per Liter of original cell culture was added. Digestion was performed at RT for 1 hour, followed by 6 washes of 4 CV Buffer B. The beads were loaded onto a gravity column and AGO protein was eluted with Buffer C (50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 300 mM imidazole, 0.5 mM TCEP). The amount of eluted AGO protein was estimated by SDS-PAGE using known concentrations of bovine serum albumin (BSA). gRNA was added to samples containing AGO protein at an estimated 1 :2 [RNA:AGO] ratio and incubated on ice for 15 min. Sample was dialyzed O/N at 4°C in the presence of TEV to cleave the His-tag. The programmed AGO protein was subjected to 2 * 5 mL HisTrap HP (Cytiva) and 2 ^/ l mL HiTrap Q FF (Cytiva) columns to remove the cleaved His-tag and excess gRNA, respectively. The homogeneous AGO protein was then purified using a modified ARPON method (Flores-Jasso 2013). Elution oligo was removed by running the RISC sample through a third HiTrap Q FF column. Flow-through was concentrated and loaded to a Superdex 200 increase 10/300 GL column (Cytiva). Protein (AGO) purity of each RISC was evaluated by SDS-PAGE, and the gRNA purity resolved on an 8 M urea, 20% (29: 1) acrylamide/bis-acrylamide denaturing gel. RISC concentrations were measured using A280. Each RISC was directly frozen in liquid nitrogen and/or diluted to 2 pM in crystallization buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM TCEP), containing 1 mg/mL BSA and 50% glycerol, before being frozen in liquid nitrogen. The RISCs were then stored at -80 °C.

Crystallization and structure determination: 1 mg/mL AGO3 : 14-nt miR-20a was incubated with a 16-nt fully complementary modified target (1 : 1.2 ratio of RISC Target) in crystallization buffer, on ice, for 3-5 min before setting up sitting-drop crystallization plates with 1 : 1 [sample:reservior] solution. Crystals grew O/N in the condition Hl 1 (0.2 M sodium citrate, 0.1 M Bis-Tris propane pH 8.5, 20% (w/v) PEG 3350) of PACT Suite (Qiagen). The condition was then optimized to 0.14 M sodium citrate, 0.1 M Bis-Tris propane pH 8.8, and 18% (w/v) PEG 3350 supplemented with 4% (v/v) 1,3 -Propanediol to grow high quality crystals. Crystals were harvested in a buffer containing 0.14 M sodium citrate, 0.1 M Bis-Tris propane pH 8.8, and 30% (w/v) PEG 3350 and flash frozen in liquid nitrogen. Crystal diffraction data were collected at the NE-CAT beamlines (Advanced Photon Source, Chicago). Molecular replacement was performed with PHASER using the crystal structure of AGO3 (PDB ID: 5VM9) as the search model. PHENIX was used for model refinement. Ramachandran statistics showed 94.57% residues in the favored region, 5.43% in the allowed region, and 0% in outlier. All figures of structures and electrostatic surfaces were generated using PyMol (www.pymol.org/) and Chimera. Target RNA extraction and labeling from crystallization drops: Crystallization samples from 8 crystallization drops (400 pL/drop) were collected and used for phenol/chloroform RNA extraction and EtOH precipitation. The extracted RNA was subsequently radiolabeled using y-³²P ATP (3,000 Ci mmol'¹; PerkinElmer) with T4 Polynucleotide kinase (NEB) at 37 °C for 1 hour, followed by 90 °C for 1 min to inactivate the kinase. The unincorporated y-³²P ATP was removed using MicroSpin™ G-25 columns (Cytiva). The RNA was then resolved on an 8 M urea, 20% (29: 1) acrylamide/bis-acrylamide denaturing gel.

Single-turnover kinetics : Target RNAs were radiolabeled using y-³²P ATP (3,000 Ci mmol'¹; PerkinElmer) with T4 Polynucleotide kinase (ThermoFisher) at 37 °C for 1 hour, followed by inactivation of the kinase at 90 °C for 1 min. Unincorporated y-³²P ATP was removed using MicroSpin™ G-25 columns (Cytiva). 2.5 nM ³²P-labeled target RNA was incubated with 10 nM RISC in 1 x Reaction Buffer (25 mM HEPES-KOH pH 7.5, 100 mM KC1, 5 mM MgCh, 5 mM DTT, 0.005% (v/v) NP-40, 0.01 mg/ml baker’s yeast tRNA, 0.05 mg/mL BSA, 0.5 U/pL Ribolock) in a total volume of 40 pL reaction at 37 °C. 5 pL of aliquots were quenched with 2 quenching dye (8 M urea, 1 mM EDTA, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromophenol blue, 10% (v/v) phenol) including 20% glycerol at 0.5, 1, 2, 3, 5, 10, and 20 min. Cleavage products were resolved on an 8 M urea, 20% (29: 1) acrylamide/bis-acrylamide denaturing gel. Phosphor images were taken by Typhoon Imager (GE Healthcare) and band intensity was quantified using Image Lab (Bio-Rad). All data were analyzed and graphed using GraphPad Prism version 9.5.0 (GraphPad Software, Inc.).

Multiple-turnover kinetics: Various concentrations of target RNA were incubated in lx Reaction Buffer with 10 nM RISC in 40 pL reactions at 37 °C. 5 pL aliquots were quenched at 0.5, 1, 1.5, 2, 2.5, 3, and 5 min with 2x quenching dye. Cleavage products were resolved on 20% denaturing gels. Phosphor images were taken by Typhoon Imager (GE Healthcare) and band intensity was quantified using Image Lab (Bio-Rad). All data were analyzed and graphed using GraphPad Prism version 9.5.0 (GraphPad Software, Inc.).

Equilibrium binding assay: The binding of RISC to target RNAs was determined using filter binding assays as previously described (Schirle et al., 2014). Specifically, 0.1 nM ³²P labeled target was incubated with increasing concentrations of RISC in 1 x Binding Buffer (25 mM HEPES-KOH pH 7.5, 100 mM KC1, 5 mM DTT, 0.005% (v/v) NP-40, 0.01 mg/mL baker’s yeast tRNA). in a total volume of 50 pL at RT for 60 min. The samples were then loaded onto a dot blot apparatus (GE Healthcare) under vacuum with the Protran nitrocellulose membrane (0.45 mm pore size, Whatman, GE Healthcare Life Sciences) on top and Hybond Nylon membrane (Amersham, GE Healthcare) at the bottom. The top nitrocellulose membrane captures RISC-target complex, while the bottom nylon membrane captures the unbound target RNA. The membranes were then washed 10 times with 75 pL of ice-cold Wash Buffer (25 mM HEPES-KOH pH 7.5, 100 mMKCl, 5 mM DTT, 0.01 mg/mL baker’ s yeast tRNA). The membranes were then air-dried, and phosphor images taken by Typhoon Imager (GE Healthcare). Signals were quantified using Image Lab (Bio-Rad). All data were analyzed and graphed using GraphPad Prism version 9.5.0 (GraphPad Software, Inc.).

Cell culture: HEK293T. A549, and HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). HCT116 cells were cultured in McCoy’s media (Gibco) supplemented with 10% FBS. All cell lines were incubated at 37 °C with 5% CO2.

Dual luciferase assay: Cells were seeded in 24-well plate with 500 pL of medium and grown up to about 90% confluency. The old media was replaced with fresh, supplemented media before the transfection. The cells were co-transfected with 50 or 150 ng of psiCHECK-2 encoding the target sequence for HEK293T and the other cell lines (A549, HeLa, and HCT116), respectively, and 6 pmol of RNA using 2.5 pL of TransIT-X2 (Minis) and 100 pL of Opti-MEM™ (Gibco). 24 hours post-transfection, cells were harvested with 500 pL of l x Phosphate Buffered Saline (PBS) per well, pelleted at 2,000 xg for 5 min. followed by cell lysis with 200 pL of Passive Lysis Buffer (PLB) per well. Centrifuge cell lysate at 21,130 x for 5 minutes and supernatant was used for dual luciferase assay. Luciferase activities were measured using GloMax® Navigator System (Promega). All luciferase emission measurements were performed using the DualLuciferase® Reporter assay (Promega). 10 pL of cell lysate was transferred to LUMITRAC™ (Greiner Bio-One) 96-well plates for luminescence recordings. 100 pL of Luciferase Assay Reagent II reagent (Promega) was added to each well to measure Flue activity. Then, the same volume of Stop & Gio® reagent (Promega) was added to measure Rluc activity. Rluc luminescence was divided by Flue luminescence, followed by normalizing to the cells transfected with only psiCHECK-2 encoding the target sequence. fa-ce// RISC assembly assay: HEK293T cells were seeded in 10 cm² plate with 10 mL of medium and grown up to about 90% confluency. The old media was replaced with fresh, supplemented media before the transfection. To confirm the RISC assembly in cells, the cells were transfected with either 5'-end radiolabeled 21-nt let-7a, 21-nt let-7a duplex, 14-nt let-7a, 14-nt let- 7a duplex, cyBR-7a, cyDR-7a, 14-nt let-7a^m, 14-nt let-7a^m duplex, cyBR-7a^m, or cyDR-7a^m using 42.5 pL of TransIT-X2 (Minis) and 1.5 mL of Opti-MEM™ (Gibco). 24 hours post-transfection, cells were harvested with 500 pL of RIPA buffer (Cell Signaling), and the cell lysates were incubated with 3 pg of anti-AG02 antibody (Wako) at RT for 2 hours, followed by incubation with Pierce™ Protein G Agarose (Thermo Scientific) beads for 2 hours at RT. Then, the beads were washed with Immunoprecipitation (IP) Wash Buffer (300 mM NaCl, 50 mM Tris-HCl pH 7.5, and 0.05% NP-40) 4 times. After removing the residual buffer, the beads were mixed with 2* quenching dye, incubated for 1 min. at 90 °C, and resolved on an 8 M urea, 20% (29: 1) acrylamide/bis-acrylamide denaturing gel. Phosphor images were taken by the Typhoon Imager (GE Healthcare).

Immunofluorescence staining: HeLa cells were grown on glass coverslips in a 6-well plate and co-transfected, using the TransIT-X2 reagent (Minis), with 200 ng of either pCAGEN-FLAG- AGO2 or pCAGEN-FLAG-AGO3 and 50 pmol of cyDR-7a carrying a 14-nt let-7a whose 3' end was conjugated with a Cy3. 24 hours post-incubation, cells were fixed with 4% paraformaldehyde in PBS for 15 min. and washed once with PBS. Cells were then permeabilized on ice with 0.5% ice-cold Triton X-100 for 6 min. The fixed and permeabilized samples were washed with PBS and blocked in a solution of 3% BSA and PBS with 0.05% Tween (PBST) for 30 min. at room temperature. A 1 :500 dilution of mouse anti -FLAG antibody (Sigma) in blocking solution was applied to the cells and incubated at 4 °C overnight. Afterward, cells were washed 3 times with PBST and incubated with a 1 :500 dilution of Alexa fluor 647 goat anti-mouse secondary antibody (Cell Signaling) for 1 hour at room temperature. Cells were washed 2 times more with PBST before a final wash with 5 ug/mL of DAPI in PBST for 10 min. After staining, the coverslips were mounted on glass microscope slides using ProLong™ Gold antifade mountant (Invitrogen). Slides were imaged with a TI2 Eclipse scanning confocal microscope (Nikon).

Data Analysis: Single and multiple-turnover kinetics. Time course data were fit to y = yo + where the initial rate, vo = Ak (Lu et al. 2003). The initial rates of target cleavage at different target concentrations were fit to Michaelis-Menten model with Prism version 9.5.0 (GraphPad Software, Inc.):

where [Nr] is total target concentration, Emax is the calculated maximum velocity, K_m is the Michaelis-Menten constant.

Dissociation constants were calculated using the following equation (6, 7) with GraphPad Prism version 9.5.0 (GraphPad Software, Inc.):

where F is fraction of target bound, Bmax is maximum number of binding sites, [Er] is total enzyme concentration, [5T] is total target concentration, and ED is the apparent equilibrium dissociation constant.

Dual luciferase assay: Relative luciferase activity was calculated by dividing the activity of Renilla luciferase by that of firefly luciferase and normalized to no-guide control. An ANOVA with Dunnett’s post-hoc test was performed to calculate statistical significance between two conditions using excel. All data were presented as mean of three replicates ± SD. *P < 0.05; **P < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Relative gene silencing was calculated by subtracting the relative luciferase activity from 1 and multiplied by 100%.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

TABLES

Table 1. Data collection and refinement statistics (molecular replacement).

Table 2. RNA oligos used in Example 3.

Table 3, Michaelis-Menten parameters for target cleavage by five different RISCs.

*Data were obtained from three independent experiments. Initial parameters were shown as mean ± SD.

Table 3 (Continued). Michaelis-Menten parameters for target cleavage by five different RISCs.

Table 4, Affinity of RISCs for targets.

*Data were obtained from three independent experiments. Kd values were shown as mean ± SD.

Table 5. Additional Sequences

Claims

CLAIMS What is claimed is:

1. A guide RNA (gRNA) comprising a cityRNA hybridized with a Booster nucleic acid, wherein the Booster nucleic acid comprises a tetranucleotide loop or a 3' 2-nucleotide overhang.

2. The gRNA of claim 1, wherein said cityRNA is 16 nucleotides or less in length.

3. The gRNA of claim 1, wherein said cityRNA is 14 nucleotides or less in length.

4. The gRNA of claim 1, 2, or 3, wherein the Booster nucleic acid hybridizes with the cityRNA.

5. The gRNA of any one of claims 1-4, wherein the Booster nucleic acid and cityRNA together form a secondary structure.

6. The gRNA of any one of claims 1-5, wherein the gRNA is greater than 18 nucleotides in length when Booster is hybridized to cityRNA.

7. The gRNA of any one of claims 1-6, wherein the Booster nucleic acid is RNA or DNA.

8. The gRNA of any one of claims 1-7, wherein the Booster nucleic acid comprises at least 2 nucleotides.

9. The gRNA of any one of claims 1-8, wherein the Booster nucleic acid comprises 25-38 nucleotides.

10. A cityRISC complex comprising a guide RNA (gRNA) and an Argonaute protein (AGO), wherein the gRNA comprises a cityRNA hybridized to a Booster nucleic acid, and wherein the Booster nucleic acid comprises a tetranucleotide loop or a 3' 2-nucleotide overhang.

11. The cityRISC complex of claim 10, wherein said cityRNA is 16 nucleotides or less in length.

12. The cityRISC complex of claim 10, wherein said cityRNA is 14 nucleotides or less in length.

13. The cityRISC complex of any one of claims 10-12, wherein the Booster nucleic acid hybridizes with the cityRNA.

14. The cityRISC complex of any one of claims 10-13, wherein the Booster nucleic acid and cityRNA together form a secondary structure.

15. The cityRISC complex of any one of claims 10-14, wherein the gRNA is greater than 18 nucleotides in length when Booster is hybridized to cityRNA.

16. The cityRISC complex of any one of claims 10-15, wherein the Booster nucleic acid is RNA or DNA.

17. The cityRISC complex of any one of claims 10-16, wherein the Booster nucleic acid comprises at least 2 nucleotides.

18. The cityRISC complex of any one of claims 10-17, wherein the Booster nucleic acid comprises 9 nucleotides.

19. A cell comprising the gRNA of any one of claims 1-9 or the cityRISC complex of any one of claims 10-18.

20. A method of regulating expression of a target nucleic acid using a cityRISC complex, wherein the cityRISC complex comprises an Argonaute protein and a guide RNA, wherein said guide RNA comprises a cityRNA hybridized with a Booster nucleic acid.

21. A method of determining a suitable Booster nucleic acid, the method comprising: identifying a cityRNA, hybridizing said cityRNA to a Booster nucleic acid, detecting whether the cityRNA hybridized with the Booster nucleic acid is more efficient at binding with an AGO or regulating gene expression, and selecting the suitable Booster nucleic acid that binds with the AGO and regulating gene expression.

22. The method of claim 20 or 21, wherein the AGO molecule comprises AGO1, AGO2, AGO3, or AGO4.

23. The method of any one of claims 20-22, wherein the target nucleic acid is RNA or DNA.

24. The method of any one of claims 20-23, wherein said cityRNA is 16 nucleotides or less in length.

25. The method of any one of claims 20-23, wherein said cityRNA is 14 nucleotides or less in length.

26. The method of any one of claims 20-25, wherein the Booster nucleic acid hybridizes with the cityRNA.

27. The method of any one of claims 20-26, wherein the Booster nucleic acid and cityRNA together form a secondary structure.

28. The method of any one of claims 20-27, wherein the guide RNA is greater than 18 nucleotides in length when Booster is hybridized to cityRNA.

29. The method of any one of claims 20-28, wherein the Booster nucleic acid is RNA or DNA.

30. The method of any one of claims 20-29, wherein the Booster nucleic acid comprises at least 2 nucleotides.

31. The method of any one of claims 20-30, wherein the Booster nucleic acid comprises 25-38 nucleotides.

32. The method of any one of claims 20-31, wherein the Booster nucleic acid protects the cityRNA from degradation by a nuclease.

33. The method of any one of claims 20-32, wherein the Booster nucleic acid improves binding the cityRNA to the AGO.