Part:BBa_K5310026

H4 hairpin

INTRODUCTION

The use of miRNAs as biomarkers and therapeutic molecules is seeing rapid advancements in the last decade. As a continuation of this, it is possible to combine the two functions with a mechanism that detects certain signature miRNAs of the disease and releases miRs with restorative effects based on a technique called Hybridization Chain Reaction.

MECHANISM OVERVIEW

The mechanism consists of two types of nucleic acid hairpins, a set of HCR hairpins and a Y-shaped one. The first HCR hairpin is activated by an initiator molecule triggering a series of unbindings and hybridizations resulting in the release of two therapeutic molecules. The Y hairpin recognizes the biomarker miRNAs and undergoes gradual unbinding of its arms upon which the initiator is released. It operates as a safety measure to make sure the HCR procedure mentioned above occurs in the right cells or tissue, where the therapeutic miRNAs should execute their function.

Different conditions are characterized by a different miRNA expression profile. It is therefore necessary to carefully select those according to which the hairpins would be designed. In the case of Multiple Sclerosis with the purpose of remyelination, one should examine which miRNAs are overexpressed in patient oligodendroglia (myelin generating cells) and which have the ability to enhance their desired function, while taking hairpin structure requirements into account. Upon literature review, discussions with neurologists and in silico testing, it was concluded that miR-125a-3p and miR-146a-5p are the biomarkers the Y hairpin would be based on, whereas miR-219-5p and miR-338-3p are the remyelinating miRNAs released by HCR.

PART FUNCTION

This part is the final of the four hairpins (H1,H2,H3,H4) participating in the Hybridization Chain Reaction. It consists of a stem (where the molecule folds and forms base pairs) complementary to H1, a loop and an exposed 5' strand that is complementary to the H2 strand that carries miR-338-3p and an H3 sequence. Once the initiator hybridizes with H1, H1 unfolds and binds to the H2 hairpin which then opens and attaches to H3, releasing miR-219. After unfolding, H3 binds with H4 that also binds to the H2 strand and releases the second miRNA, thus completing the cycle and achieving therapeutic results. The hybridizations, loosen base pair bonds on the stem, allowing it to bind to the H1 sequence and activate it like the initiator. The reaction carries on like this, continuing to produce miRNAs until the hairpin supply is exhausted.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 46
  Illegal EcoRI site found at 69
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 46
  Illegal EcoRI site found at 69
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 46
  Illegal EcoRI site found at 69
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 46
  Illegal EcoRI site found at 69
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 46
  Illegal EcoRI site found at 69
  
• 1000
  COMPATIBLE WITH RFC[1000]