Ribozymes and Deoxyribozymes

Nucleic Acid Chemistry

Third term 2022-2023

Randy Bryant

Department of Biochemistry and Molecular Biology

Johns Hopkins Bloomberg School of Public Health

Catalytic RNA

Discovered by Thomas Cech and

colleagues in 1982

Was a 413 base pair intron (IVS) located

in the 26 S ribosomal RNA gene of
Tetrahymena thermophila

Underwent a self-splicing reaction in the

presence of monovalent and divalent
cations, and a guanosine cofactor

Required no proteins!
 Ribozymes

Because the IVS RNA is not an enzyme

but has some enzyme-like characteristics,
we call it a ribozyme, an RNA molecule
that has the intrinsic ability to break and
form covalent bonds.

Kruger et. al., “Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena” Cell 31, 147 (1982)
 Group I self-splicing introns

Are the most abundant class of self-splicing introns

Have been found in algae, lichen, fungi, and bacteria

Are found in protein, tRNA , and rRNA genes

Require a guanosine cofactor to initiate the self-splicing

reaction:

guanosine, GMP, GDP, or GTP

 Group I intron self-splicing reaction

guanosine
cofactor
G-OH
exon I intron exon II
5´- UpA GpU -3´

The 3´OH group of guanosine

attacks the phosphorus at the
5´ splice site

exon I intron exon II

5´- U-OH -3´ 5´- GpA GpU -3´

The 3´OH group of exon I

attacks the phosphorus at
the 3´ splice site

intron
5´- GpA G-OH -3´ released intron

exon I exon II
5´- UpU -3´ spliced exons
 Tetrahymena group I intron structure

cleavage site

X-ray crystal structure secondary structural organization

Golden et. al, “A preorganized active site in the crystal structure of the Tetrahymena ribozyme” Science 282, 259 (1998)
Functional organization of the Tetrahymena group I intron

guanosine binding terminal G

site
3´-exon
ω
5´-exon

-1 1

cleavage site
22

intron
internal guide
sequence conserved G:U
base pair
(wobble)

Adapted from: Biochemistry, 6th ed. Freeman 2007

Three-metal ion mechanism for Tetrahymena group I intron cleavage

G:U base pair Metal A: interacts with the 3´-OH

of the 5´-exon

Metal B: interacts with the 3´-OH

of the guanosine cofactor
internal guide
sequence
Metal C: interacts with the 2´-OH
2´ 3´ of the guanosine cofactor
and an oxygen of the
phosphodiester bond
guanosine
cofactor Metals = Mg2+

Doudna and Cech, “The chemical repertoire of natural ribozymes” Nature 416, 222 (2002)
Tetrahymena group I intron reaction sequence

Adapted from: Biochemistry, 6th ed. Freeman 2007

Re-engineered Tetrahymena ribozymes

Prepared a modified version of

the Tetrahymena intron (1986)

Deleted 19 nts from 5´-end and

deleted the terminal G residue
from 3´-end: L-19 IVS RNA

Functioned as a sequence-specific
RNA endonuclease
Conversion of Tetrahymena group I intron into an RNA endonuclease

cleaved
external RNA external RNA

Endonuclease activity of L-19 IVS is an intermolecular version of the

first step of the self-splicing reaction

Zaug, et al., “The Tetrahymena ribozyme acts like an RNA restriction endonuclease” Nature 324, 429 (1986))
Conversion of Tetrahymena group I intron into an RNA endonuclease

cleaved
external RNA external RNA

Endonuclease activity of L-19 IVS is an intermolecular version of the

first step of the self-splicing reaction

Zaug, et al., “The Tetrahymena ribozyme acts like an RNA restriction endonuclease” Nature 324, 429 (1986))
Sequence-specific Tetrahymena L-19 endonucleases

Generated a series of Tetrahymena

L-19 ribozymes (1989)

Each variant had a different

internal guide sequence

Each variant cleaved RNA substrates

in a sequence-specific manner
 Sequence-specific Tetrahymena L-19 endonucleases

L-19 endonuclease

RNA substrate

conserved cleaved
G:U base pair RNA products
internal guide
sequence

Varied RNA cleavage specificity

by changing the nucleotides in
the internal guide sequence

Murphy and Cech, “Alteration of substrate specificity for the endonucleolytic cleavage of RNA by the Tetrahymena ribozyme” PNAS 86, 9218 (1989)
Sequence-specific cleavage of RNA by variant L-19 ribozymes

Each L-19 variant cleaved the predicted RNA substrate in a

sequence-specific manner

Murphy and Cech, “Alteration of substrate specificity for the endonucleolytic cleavage of RNA by the Tetrahymena ribozyme” PNAS 86, 9218 (1989)
RNA-cleaving ribozymes
 Self-cleaving ribozymes

Are five classes of naturally-occurring self-cleaving ribozymes

Hammerhead
Hairpin
Hepatitis delta virus (HDV)
Varkud satellite (VS)
GlmS

Have different secondary and tertiary structures

But catalyze the same chemical reaction using similar

mechanisms
Naturally-occurring self-cleaving ribozymes

Cochrane and Strobel, “Catalytic stategies of self-cleaving ribozymes” Accounts Chem. Res. 41, 1027 (2008)
General reaction mechanism for self-cleaving ribozymes

3´ 2´
2´,3´-cyclic phosphate
cleavage fragment
5´

5´-hydroxyl
cleavage fragment

cleavage site transition state cleavage products

Ferre-D’Amare and Scott, “Small self-cleaving ribozymes” Cold Spring Harb.Perspect. Biol. 2:a003574 (2010)
 Hammerhead RNA

First self-cleaving RNA to be discovered (1986)

First catalytic RNA X-ray crystal structure (1995)

Smallest catalytic RNA (typically 50-150 nucleotides)

Most extensively studied self-cleaving RNA

Considered to be the prototypical ribozyme

Self-cleavage of hammerhead RNA sequences

Rolling circle replication of plant pathogen

RNA (various satellite and viroid RNAs)

Self-cleavage of concatemeric RNA to

produce monomeric products

Hammerhead RNA sequence

Scott, “What can new hammerhead ribozyme structures teach us about design?” RNA Technologies and Their Applications (2010)
 Hammerhead RNA structure

Peach Latent Mosaic Viroid

Hammerhead RNA

secondary structure folded structure X-ray crystal structure

Flores, et. al., “Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs”, Front. Microbiol. 18 June 2012
 Naturally-occurring hammerhead RNAs

Are three types of hammerhead RNAs, which differ in the location of

the open stem of the sequence

All have a similar Y-shaped fold and a conserved catalytic core

Hammann, et. al., “The ubiquitous hammerhead ribozyme”, RNA 18, 871-875 (2012)
 Hammerhead RNA catalytic core

Hammerhead RNAs have a 17-nucleotide

cleavage catalytic core

Includes 13 invariant nucleotides

Self-cleavage reaction occurs between

residue 1.1 and C-17

Scott, “What can new hammerhead ribozyme structures teach us about design?” RNA Technologies and Their Applications (2010)
 Hammerhead RNA active site structure

cleavage
2´
1

5´
2´

3´

catalytic core X-ray crystal structure

Scott, “What can new hammerhead ribozyme structures teach us about design?” RNA Technologies and Their Applications (2010)
 Trans-acting hammerhead ribozymes

cleavage cleavage
17
1.1 3´

5´

full-length HHR bimolecular construct

Hammerhead RNAs have been re-engineered as bimolecular

constructs
Can be used to catalyze trans-cleavage of RNA substrates

Adapted from: Flores, et. al., “Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs”, Front. Microbiol. 18 June 2012
Design strategy for trans-acting hammerhead ribozymes

RNA target

trans-acting
HHR

NUH: cleavage site on the target RNA sequence (H17 = C, U, or A)

Stems I and III: bind RNA target sequence
Stem II: connects separate parts of the catalytic core

Tedeschi et. al., “Hammerhead ribozymes in therapeutic target discovery and validation” Drug Discovery Today 14, 776 (2009)
 RNA cleavage by trans-acting hammerhead ribozymes

cleaved
RNA target RNA target

trans-acting HHR-target cleavage release

HHR complex

Can be used for the sequence-specific cleavage of RNA targets

Research applications of hammerhead ribozymes

Tedeschi et. al., “Hammerhead ribozymes in therapeutic target discovery and validation” Drug Discovery Today 14, 776 (2009)
Artificial ribozymes
Artificial self-cleaving ribozymes

Breaker and colleagues used in vitro selection

to generate artificial self-cleaving RNAs (2000)

Sought to identify new ribozymes with

enhanced catalytic properties compared
to naturally-occurring ribozymes

Could be used as sequence-specific RNA

cleavage agents for gene inactivation
applications
 In vitro selection for self-cleaving ribozymes

Initial RNA construct:

random nucleotide
sequences

potential catalytic potential cleavage sites

domain

Generated a pool of ~1014 different RNAs

Tang and Breaker, “Structural diversity of self-cleaving ribozymes” PNAS 97, 5784 (2000)
In vitro selection for self-cleaving ribozymes

Incubated RNA pool under permissive conditions

(pH 7.5, 20 mM MgCl2; 4 hr at 23 oC)

Isolated the 5´-cleavage fragments by PAGE

These fragments corresponded to those RNA

molecules that had undergone self-cleavage

Amplified cleavage fragments by RT-PCR, using

a template that restored the nucleotides lost by
cleavage
*
Coverted amplified dsDNA to ssRNA using T7 RNA
polymerase

Repeated procedure to enrich pool for Mg2+-dependent

self-cleaving RNA molecules

Tang and Breaker, “Structural diversity of self-cleaving ribozymes” PNAS 97, 5784 (2000)
 Artificial self-cleaving ribozymes

Identified 12 distinct classes of self-cleaving RNA molecules

Tang and Breaker, “Structural diversity of self-cleaving ribozymes” PNAS 97, 5784 (2000)
 X motif ribozyme

Dominant ribozyme after 25 rounds of

in vitro selection

Consists of four double-helical stems

cleavage site
arranged in an X shape
IV I
Undergoes self-cleavage in the presence
III
II of Mg2+, specifically at an unpaired G
residue

Produces 2´,3´-cyclic phosphate and

5´-hydroxyl termini at cleavage site

Consistent with a Mg2+-dependent cyclization mechanism

Tang and Breaker, “Structural diversity of self-cleaving ribozymes” PNAS 97, 5784 (2000)
 X motif ribozyme

cleavage site

cleavage site

IV I

II
III

X motif ribozyme bimolecular construct

Showed that the X motif ribozyme could be reorganized as

a 43-nt enzyme component and 21-nt substrate component

Tang and Breaker, “Structural diversity of self-cleaving ribozymes” PNAS 97, 5784 (2000)
 Site-specific RNA cleavage by X motif ribozymes

32P

5´-32P- labeled products

Generated three variants of the X motif ribozyme that differed in

the base-pairing potential of arms I and IV (X-E1, X-E2, and X-E3)

Each catalyzed the site-specific cleavage of an RNA substrate

Tang and Breaker, “Structural diversity of self-cleaving ribozymes” PNAS 97, 5784 (2000)
Deoxyribozymes
 Potential advantages to deoxyribozymes

DNA is more stable than RNA, due to the absence of a 2´-OH group:

RNA DNA
5´RNA 5´DNA

cyclization no cyclization

3´RNA 3´DNA

DNA is also easier and less expensive than RNA to synthesize

 Deoxyribozymes

No naturally-occurring DNAs with catalytic activity have

been identified

However, DNAs with catalytic activity have been generated

by in vitro selection

Referred to as deoxyribozymes, DNA enzymes, or DNAzymes

Catalytic DNA molecules

Catalytically-active DNA molecules were

first described by Joyce and colleagues
in 1995-1997

Used in vitro selection to generate

DNA molecules that could catalyze
RNA cleavage
 Selection criteria for RNA-cleaving DNA enzymes

1. Ability to cleave RNA with multiple turnovers under simulated

physiological conditions (2 mM MgCl2/150 mM KCl, pH 7.5, 37°C)

2. Ability to recognize the RNA substrate through Watson-Crick

base pairing

3. Generalizability to other RNA substrates by changing the sequence

of the substrate-recognition domain

4. Catalytic efficiency meeting or exceeding that of comparable RNA

enzymes

5. Total composition of no more than 50 deoxyribonucleotides (and

preferably fewer)

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
 In vitro selection of RNA-cleaving DNA enzymes

Initial chimeric construct:

potential cleavage sites

(RNA)
biotin tag (for attachment to
streptavidin column)

random nucleotide sequences = potential catalytic domain

(DNA)

Generated a pool of ~1014 different chimeric molecules

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
 In vitro selection of RNA-cleaving DNA enzymes

7 1
Applied chimeric pool to a streptavidin
column
Eluted column with a solution containing
10 mM MgCl2 at pH 7.5 and 37 oC
Recovered released fragments

These fragments corresponded to those

molecules that had undergone self-cleavage

Amplified fragments by PCR, restoring the

biotin tag and the target ribonucleotide
sequence

Repeated procedure to enrich pool for Mg2+-dependent RNA-cleaving

DNA molecules

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
 RNA-cleaving DNA enzymes

cleavage cleavage

RNA RNA

DNA DNA

Y = U or C
R = A or G

8-17 DNA enzyme 10-23 DNA enzyme

(17thclone from round 8) (23rd clone from round 10)

Identified two promising RNA-cleaving DNA molecules

And showed they could be reorganized as bimolecular constructs

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
 Structural organization of the 10-23 DNA enzyme

cleavage

RNA substrate

DNA enzyme
substrate substrate
recognition recognition

catalytic domain

Consists of a catalytic domain (15-nucleotides) and two substrate recognition

domains (7 nucleotides each)

Catalyzes a Mg2+-dependent cleavage between an unpaired purine (R= A or G)

and a paired pyrimidine (Y = U or C) of an RNA substrate

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
 RNA cleavage by the 10-23 DNA enzyme

Designed a 10-23 DNA enzyme to cleave an

RNA substrate at the target sequence:
5´-32P-labeled
RNA substrate
5´-GGAGAGAGA-UGGGUGCG-3´ translation initiation region
HIV-1 gag/pol mRNA

5´-32P-labeled Produced 2´,3´-cyclic phosphate and 5´-hydroxyl

RNA product
terminated cleavage products

Consistent with a Mg2+-dependent

cyclization mechanism

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
10-23 DNA enzyme follows Michaelis-Menten kinetics

[10-23 DNA] = 0.004 nM (fixed)

[17-mer RNA substrate] = 0.02- 4 nM

5´-GGAGAGAGA-UGGGUGCG-3´

cleaved
RNA target RNA target

10-23 DNA

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
Catalytic activity of various RNA-cleaving enzymes

Santoro and Joyce, “A general purpose RNA-cleaving DNA enzyme” PNAS 94, 4262 (1997)
 Deoxyribozyme Dz13

AP-1 transcription factors participate in oncogenic transformation,

angiogenesis, dysregulation of tumor-cell proliferation and apoptosis,
invasive growth, and metastasis

c-JUN is a protypical member of the AP-1 family of transcription factors

c-JUN expression is minimal in normal adult human tissues, but is

increased in various pathologies and in abnormal tissue conditions,
including basal-cell carcinoma, which makes it a potential therapeutic
target

Dz13 is a DNAzyme that specifically targets human c-JUN messenger RNA

 Deoxyribozyme Dz13

target site

34 nt

time course

Dz13 cleaves human c-Jun mRNA in

a sequence-dependent manner

concentration
dependence

Khachigian, et.al., “c-Jun regulates vascular smooth muscle cell growth and neointima formation after arterial injury. Inhibition by a novel DNA enzyme targeting c-Jun” J. Biol. Chem. 277, 22985 (2002)
Deoxyribozyme Dz13

2013
RNA-cleaving ribozymes and deoxyribozymes

Breaker, “Natural and engineered nucleic acids as tools to explore biology” Nature 432, 838 (2004)
 DNA-cleaving deoxyribozymes

DNA-cleaving ribozymes or deoxyribozymes are a challenge:

RNA DNA
5´RNA 5´DNA

cyclization no cyclization

3´RNA 3´DNA

DNA does not have a 2´-OH group to initiate a phosphodiester bond

cleavage reaction
DNA-cleaving deoxyribozymes

Breaker and colleagues used in vitro selection

to generate DNA molecules that could cleave
DNA substrates (2013)

Identified two classes of Zn2+-dependent

self-cleaving deoxyribozymes
 DNA-cleaving deoxyribozymes

*
*

15 nt catalytic core 36 nt catalytic core

Identified two classes of Zn2+-dependent self-cleaving deoxyribozymes

Gu, et.al., “Small, highly active DNAs that hydrolyze DNA” J. Am. Chem. Soc. 135, 9121 (2013)
 DNA-cleaving deoxyribozymes

cleavage site

kcat ~ 1 min-1

Class I
bimolecular construct
1012-fold faster than uncatalyzed reaction

Showed that the DNA-cleaving deoxyribozymes could function as

bimolecular constructs
Gu, et.al., “Small, highly active DNAs that hydrolyze DNA” J. Am. Chem. Soc. 135, 9121 (2013)
 DNA-cleaving deoxyribozymes

cleavage
3´
5´

Class I
mass spectroscopic analysis
bimolecular construct

I-R3 produces 5´-phosphoryl and 3´-hydroxyl cleavage products

So cleavage reaction follows a true phosphodiester bond hydrolysis mechanism

Gu, et.al., “Small, highly active DNAs that hydrolyze DNA” J. Am. Chem. Soc. 135, 9121 (2013)
 Artificial ribozymes

Breaker and Joyce, “The expanding view of RNA and DNA function” Chem. Biol. 21, 1059 (2014)