Sequence-Specific Gene control

Amino acid – Base interactions

Hoogsteen faces

Phage Display

phage: holds DNA inside, expresses proteins outside

DNA-binding experiment

Recognition Code of Zinc fingers

what locations and residues on zinc fingers bind to DNA

Zinc Finger DNA cleaver

A non-specific nuclease (FokI) borrowed from restriction enzymes is attached to the Zn-Finger DBD; artificial restriction enzymes

FokI

non-specific nuclease; cleaves phosphodiester groups 9 bp away on the 5′ strand and 13 bp away on the 3′ strand, as indicated by arrows.

Activation

turns transcription on

Repression

turns transcriptions off

Transcription regulation logic

logic gates

KRAB (Krüppel-associated box)

attached to sequence-specific zinc finger → recruits HMTs → trimethylate histones → silence genes

approximately one third (290) of the 799 different zinc-finger proteins present in the human genome; the largest single family of transcriptional regulators in mammals

Virus Protein 16 (VP16)

transcription factor from Herpes simplex virus-1 (HSV-1); One of the strongest activation domains known; fused to a DNA-binding domain (DBD) of another protein in order create a sequence-specific artificial transcription activator; facilitates assembly of the preinitiation complex

Zinc finger regulator

add effector domain for site-specfic regulation

CRISPR/Cas9

plasmid used to alter genome but can be engineered to remove nuclease activity to add regulatory activity

less specific than zinc fingers; but easier to deliver

TALENs (Transcription activator-like effector nuclease)

engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations

sequence-specific targeting issue

Specific targeting of DNA requires recognition of long stretches of DNA; But large proteins and DNA are generally not cell permeable

Small Molecule DNA Binders

intercalators

minor groove binders

major groove binders

Distamycin/Netropsin

minor groove binders driven by hydrophobic effect

read A-T rich sequences: more flexible, pi-stacking

2:1 dimer stack: possibility to engineer specificity

Rise per Residue of Polyamide Is Similar to Rise of B-DNA

Altered distamycin - C-G/G-C specificity

Imidazole/Pyrrole targets G*C

Pyrrole/Imidazole targets C*G

Pyrrole/Pyrrole targets A*T and T*A

Altered distamycin - lower needed concentration

hairpin polyamide motif

Kd = 14 nM

makes molecule larger

footprinting experiment

where does this molecule bind to DNA?

DNAse randomly cutes DNA but can’t cut where DNA is bound → white space on gel

concentration dependent: 50% concentration = Ka

gel shift experiment

NATIVE gel electrophoresis, not denaturing - double strand

finds binding affinity - concentration dependent

molecule binding motifs and affinity

Altered distamycin - A-T vs T-A discrimination

use hydroxy pyrole

Downregulation of HIV Transcription

hairpin binders prevent binding to transciptional zones

Chlorambucil

mustard gas reactivity

animal chemotherapeutic

Regulation by HDAC Inhibitor

Friedreich’s Ataxia

an inherited neurodegenerative disorder that impacts children

triple-repeat neurological disorder → repeats cause folding, more heterochromatin, and stalled transcription → less mRNA made

BRD4

passive scaffold protein and active kinase that phosphorylates RNA polymerase II

possible therapeutic for Friedreich’s Ataxia → turn on transcription