CH391L/S13/In vitro Selection of FNAs

From OpenWetWare

Jump to: navigation, search


Contents

Introduction

Although the vast majority of such ligand binding activities or enzymatic activities known are performed by proteins, a secondary subset of these fall under the category of Functional nucleic acids (FNAs). FNAs are RNA, ssDNA, or XNA(nucleic acid analogues) that perform an activity such as binding or catalyzing a reaction. FNAs are grouped into three main categories Aptamers, Ribozymes, and Deoxyribozymes that are further classified into either natural or artificial depending on their origin. The exception being Deoxyribozymes as they have yet to be discovered in a living organism. Still, It was only in the 1980s that the 1st ribozyme was discovered that we started to study FNAs and have allowed for the discovery of new methods, such as the SELEX or In vitro selection process that we are expanding their potential both as tools for exploring biology and real world problem solving.

Note to self: mention RNA World

Functional Nucleic Acids

[1],[2],[3],[4][5]

Ribozymes

As previously mentioned ribozymes fall under the category of enzymes. Most of the ribozymes studied up until recently in living organism fall into 9 classes. Of these most perform some type scission and ligation reaction. In the case of in vitro selected ribozymes their function has been expanded to include . It was in such attempts that TPP ribozyme biobrick was made from both by combining an aptamer and a ribozyme for regulatory purposes. Most of other focus also on the regulation of an specific part, the self-modification of the part itself or both. Finally, one of the newest tools available to new tool are flexizymes that perform a self-aminoacylating reaction on an in vitro selected tRNA with a N70 region and that can add nonnatural amino acids by reprogramming genetic code[6].

Natural Ribozymes [7]

  • Cleaving Ribozymes
      1. 1 Self-cleaving i.e. The Hammerhead family (satellite RNA)
      2. 2 Trans-cleaving i.e. RNase P
  • Splicing Ribozymes
      1. 3 Group I
      2. 4 Group II
  • Riboswitches
      1. 5 Thermosensors
      2. 6 sRNAs
      3. 7 T-Boxes
      4. 8 Metabolites
      5. 9 Magnesium

Deoxyribozymes

An interesting discovery was made in the early 1990s, when for the first time DNA was shown that, besides being a genetic information storage molecule, it could also be both an enzyme and an aptamer. In the figure below you can observe 8-17, an RNA cleaving DNA enzyme. This molecule with 10-23 were the first to be described and tested in vivo as potential new therapies for cleaving the expressed mRNA of a virus. [5] Although proteins offer a larger diversity chemistries, depending on amino acids vs. both kinds of nucleic acids, as the latter ones depend on a limited array of nucleotides. Until know around a dozen distinct types of reactions. These include the following activities such as self-phosphorylation, RNA labeling, depurination,etc [8]

Image:8-17OP.png


Aptamers and Riboswitches

The word aptamer from the latin aptus and translates as the past participle of to fit were originally identified by employing the protocol SELEX. Therefore the word Aptamer describes their basic function as RNA or single stranded DNA (ssDNA)that can bind a ligand by assuming an specific structure.[9, 10] Yet, it would take several years until the discovery of the first in vivo aptamer or riboswitch [11]. See the following page to get a better understanding of aptamers and riboswitches.

In vitro Selection of Functional Nucleic Acids


The image presented describes the basic method for performing a SELEX or In vivo selection experiment using single stranded nucleic acids (RNA,ssDNA,XNA) that are chemically synthesizedan have a constant region (CR) and a random region. Having the CR allows later amplification using PCR. The first step is subjecting the population of single stranded nucleic acids to specific selective condition in which function is possible. Then a (2) diverse subset of the population will perform the desired function and will be then (3) PCR amplified to make double stranded nucleic acids with the use of the CR introduced previously. The previous step is necessary for Therefore the selection can continue to a following round, while at the same time a sample is obtained and can be sequenced. [12]

New Methods that assit In vitro selections

Computational Methods

  • Mfold - originally developed as a GUI or graphical user interface server for the prediction of the secondary structure of single stranded nucleic acids. The core algorithm predicts structures based on minimum free energies necessary to fold a structure. This algorithm can be used initially as a step to predict possible secondary structures of active sequences [13].

FRET

  • Fluorescent analogues

8-17 fluorescent probe and quencher pair that allows measuring activity of the population of molecules. This has been also dome for ribozymes.citeLi

Next Generation Sequencing

Take the PCR amplified molecules at every round and allows to trace the in vitro selection portion of the population that performed the desired activity.

References

  1. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, and Cech TR. . pmid:6297745. PubMed HubMed [Cech1982]
  2. Guerrier-Takada C, Gardiner K, Marsh T, Pace N, and Altman S. . pmid:6197186. PubMed HubMed [Altman1983]
  3. Ellington AD and Szostak JW. . pmid:1697402. PubMed HubMed [Ellington1990]
  4. Tuerk C and Gold L. . pmid:2200121. PubMed HubMed [Gold1990]
  5. Murakami H, Ohta A, Goto Y, Sako Y, and Suga H. . pmid:17150804. PubMed HubMed [GoTo2006]
  6. Serganov A and Patel DJ. . pmid:17846637. PubMed HubMed [Patel2007]
  7. Breaker RR. . pmid:11187837. PubMed HubMed [Breaker2000]
  8. isbn:978-0-387-73711-9. [Silverman2009]
  9. Wilson DS and Szostak JW. . pmid:10872462. PubMed HubMed [Wilson1999]
  10. Winkler W, Nahvi A, and Breaker RR. . pmid:12410317. PubMed HubMed [Winkler2002]
  11. Emilsson GM and Breaker RR. . pmid:12022469. PubMed HubMed [Breaker2002]
  12. Zuker M. . pmid:12824337. PubMed HubMed [Zucker2003]
All Medline abstracts: PubMed HubMed
Personal tools