Difference between revisions of "Part:BBa K3407006"

Revision as of 12:14, 26 October 2020

Short hairpin RNA (shRNA): a dmDicer-2 substrate and potential trigger of RNAi.

Usage and Biology

shRNAs are single RNA molecules with a strong, self-assembling secondary structure and a loop. In the scientific community, RNA interference (RNAi) terminology can be confusing by referring to different molecules with the same name. The diverse nature of RNA structures makes it difficult to reflect its heterogeneity while keeping a minimum number of terms. Double-stranded RNA (dsRNA) is commonly referred to as complementary sequences of RNA, that hybridise to form a fragment of dsRNA of about 200-600bp [1][2][3][4][5]; others use the term to name shRNAs [6], while some refer to shRNAs as such [7][8]. The reality is, many structures of single-stranded RNAs (ssRNAs) can hybridise with others or themselves to become fully double-stranded, or reserve only a specific part of the whole to form dsRNA.

RNAi is triggered when dsRNA is present in the cytoplasm of most eukaryotes. Dicer is the enzyme in charge of recognising and cleaving dsRNA into either micro RNA (miRNA), or small interfering RNA (siRNA) [9]. In eukaryotes other than plants, siRNA arise from the processing of dsRNA without mismatches, while miRNA are mainly thought to come from mismatched dsRNA processing. In insects, miRNAs are a product of Dicer-1 processing, while siRNA are generated by Dicer-2 [10], at least the latter in a ATP-independent manner [11]. One strand of these small RNAs is selected and loaded in RNA Induced Silencing Complex (RISC), to direct post-transcriptional gene silencing. It is attained by interfering with mRNAs that show a certain degree of complementarity with the selected strand from miRNAs, or by cleaving the mRNA if it shows 100% complementarity with the selected strand from siRNA [12]. This statements remains partially true, as crossovers between the two pathways have been shown to happen, as siRNA can behave like miRNA by interfering with mRNAs with only a certain degree of complementarity, corresponding to a 100% match only with the “seed region” (2-8 nt) of the loaded strand in RISC [13].

Figure 1: Illustrated representation of Dicer-2 from D. melanogaster (also named dmDicer-2) processing of shRNA into siRNA.

RNA hairpins can take both pathways, leading to either siRNA or miRNA. To produce miRNA, a transcript called pri-miRNA is processed by Drosha-DGCR8 complex into pre-miRNA in the nucleus, to later be exported to the cytoplasm by Exportin-5 [14]. To do so, this latter protein needs to recognise the loop of the pre-miRNA and its overhang [15]. Next, pre-miRNA is further processed by Dicer to produce miRNAs [16][17]. On the other hand, when short RNA hairpins do not show any mismatches in their dsRNA sequence, they can be processed by Dicer-2 to produce siRNA [18], as depicted in Figure 1. Interestingly, shRNA can bypass Dicer processing to perform siRNA-like silencing by directly loading into RISC [19]. Therefore, shRNAs are valuable candidates to mediate specific gene silencing through siRNA pathway.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Experimental results

shRNA can be transcribed with T7 RNA polymerase

shRNA are produced in vitro using the T7 RiboMAX RNAi express kit from Promega, which makes use of the viral T7 RNA polymerase.

Figure 2: Schematic representation of the shRNA in vitro production using T7 RiboMAX kit from Promega. Primer pairs are annealed in the T4 ligase buffer by heating at 95ºC for 5 minutes and left cooling at room temperature for 30 minutes. Once template is annealed, T7 RNA polymerase is added to transcribe the DNA template at 42ºC for 2 hours. shRNAs self assemble as soon as they are produced. (B) DNA template design for in vitro shRNA production. T7 promoter followed by eGFP target sequences with inverted repeats are linked by the loop region, and finally ends with a GG overhang.

For each short hairpin, a pair of complementary primers were annealed to form a DNA template for transcription (Figure 2B). The DNA template was designed to possess a T7 promoter followed by a 27nt inverted repeat sequence taken from the eGFP gene (nt 78 to 105), and linked together by the 9nt sequence containing the target of Fox-1 RBD. On the 3’ termini, two GG were added to produce the desired overhang (BBa_K3407022). This leads to a successful transcription without the need to use a terminator (Figure 2A).

[[File:T--TUDelft--RiboMAX.jpeg|thumb|none|800px|Figure 2: Schematic representation of the shRNA in vitro production using T7 RiboMAX kit from Promega. Primer pairs are annealed in the T4 ligase buffer by heating at 95ºC for 5 minutes and left cooling at room temperature for 30 minutes. Once template is annealed, T7 RNA polymerase is added to transcribe the DNA template at 42ºC for 2 hours. shRNAs self assemble as soon as they are produced. (B) DNA template design for in vitro shRNA production. T7 promoter followed by eGFP target sequences with inverted repeats are linked by the loop region, and finally ends with a GG overhang.]]

==a== shRNA (BBa_K3407022), shRNA* (BBa_K3407023) ===References=== Ordered List

@@ Line 33: / Line 33: @@
 For each short hairpin, a pair of complementary primers were annealed to form a DNA template for transcription (Figure 2B). The DNA template was designed to possess a T7 promoter followed by a 27nt inverted repeat sequence taken from the eGFP gene (nt 78 to 105), and linked together by the 9nt sequence containing the target of Fox-1 RBD. On the 3’ termini, two GG were added to produce the desired overhang (<html><a href="https://parts.igem.org/Part:BBa_K3407002" target="_blank"><b>BBa_K3407022</b></a><html>). This leads to a successful transcription without the need to use a terminator (Figure 2A).
+<div><ul>
+<center>
+   <li style="display: inline-block;"> [[File:T--TUDelft--RiboMAX.jpeg|thumb|none|800px|<b>Figure 2:</b> Schematic representation of the shRNA in vitro production using T7 RiboMAX kit from Promega. Primer pairs are annealed in the T4 ligase buffer by heating at 95ºC for 5 minutes and left cooling at room temperature for 30 minutes. Once template is annealed, T7 RNA polymerase is added to transcribe the DNA template at 42ºC for 2 hours. shRNAs self assemble as soon as they are produced. (B) DNA template design for in vitro shRNA production. T7 promoter followed by eGFP target sequences with inverted repeats are linked by the loop region, and finally ends with a GG overhang.]] </li>
+</center>
+    </ul></div>
+==a==
+shRNA (<html><a href="https://parts.igem.org/Part:BBa_K3407022" target="_blank"><b>BBa_K3407022</b></a><html>), shRNA* (<html><a href="https://parts.igem.org/Part:BBa_K3407023" target="_blank"><b>BBa_K3407023</b></a><html>)
 ===References===