The document contains summaries of 6 topics from a nanobiology mid-term exam: 1) CRISPR-Cas system uses RNA and Cas9 enzyme to cut viral DNA that has invaded bacteria. Researchers now use CRISPR to edit genes. 2) mRNA vaccines introduce mRNA that codes for a disease antigen, getting the body to produce and respond to the antigen. 3) FET biosensors use a gate to control current flow through a semiconductor, and can detect biomolecules by their effect on the gate. 4) LAMP amplification detects DNA through strand displacement and forms stem-loop structures without thermocycling. 5) CAR-T therapy attaches antigen receptors to T cells to target

1. Nanobiology Mid-Term Exam (10/19/2021)
Student ID No.: 02013035 Student Name: Mesele Tilahun Belete
1. Explain how CRISPR-Cas system works.
CRISPR- stands for "clustered regularly interspaced short palindromic repeats. CRISPR-Cas is an
adaptive immune system. When bacteria are infected by a virus, they use their CRISPR system to cut
up the invading viral DNAand insert pieces of it (spacers) into their own genome as a memory of the
infection. They transcribe the spacers into RNA, which can form a complex with the Cas9 enzyme.
These complexes monitor the cell for any DNAsequence complementary to the RNA.If matching (viral)
DNA is encountered, the spacer RNA-Cas9 complex binds to it and cuts the viral DNAto prevent it
from replicating and destroy the invader's genome. Researchers are now building different versions of
CRISPR RNAs. These lab-made RNAs guide the enzyme to cut specific genes in other organisms.
Researchers use them, like a genetic scissor, to edit or alter specific genes so that they can then study
how the gene works, repair damage to broken genes, insert new genes or disable harmful ones.
2. Explain how mRNAvaccine works.
RNAvaccines utilize a different method, using the process through which cells produce proteins: cells
use DNA as the template to make messenger RNA (mRNA) molecules, which are then translated to
build proteins. RNAvaccines work by introducing an mRNAsequence that coded for a disease specific
antigen, once produced within the body, the antigen is recognized by the immune system, preparing it
to fight the real thing.
3. Explain how Field-Effect Transistor (FET) biosensor works.
Afield-effect transistor (FET) consists of a channel of N- or P-type semiconductor material through
which current can flow, with a different material (laid across a section of the channel) controlling the
conductivity of the channel. One end of the channel is known asthe source, the other end of the channel
is called the drain, and the control mechanism is called the gate. By applying a voltage to the gate, you
control the flow of current from the source to the drain. Leads are attached to the source, drain, and
gate. The two common families of FETs, the junction FET (JFET) and the metal oxide semiconductor
FET (MOSFET) differ in the way the gate contact is made on the source-drain channel. The gate-
channel junction of the JFET must always be reverse biased otherwise it may behave as a diode. In the
MOSFET the gate-channel contact is a metal electrode separated from the channel by a thin layer of
insulating oxide.
4. Explain how LAMP works.
LAMP - stands for Loop mediated isothermal amplification that is an isothermal nucleic acid
amplification technique. In contrast to PCR, The LAMP reaction proceeds at constant temperature and
does not require thermos cycler (No DNAdenaturation, primer annealing and elongation). Through the
use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene, the
reaction process proceeds ~65o
c using strand displacement reaction. In the target gene, the B1, B2, and
B3 regions at the 5-side and the F3c, F2c, and F1c regions at the 3’-side are the distinct regions.
Amplification and detection of gene completed in single step, by incubating the mixture of samples,
primers, DNA polymerase with strand displacement activity and substrate at constant temperature
(~65o
c). There is no need for a step to denature double stranded into a single stranded form. Because of
its high specificity, the presence of amplified product can indicate the presence of the target gene. The
detection can be done at end point at naked eye, based on turbidity and color. All primer activities carry
out based on the following summarized steps
1) One of the LAMP primers can anneal to complimentary sequence of double stranded target
DNA, the initiates DNA synthesis using DNA polymerase with strand displacement activity,
displacing and releasing a single stranded DNA. With LAMP method, unlike with PCR, there
is no need for heat denaturation of double stranded DNAinto a single strand.
2) Through the activity of polymerase with strand displacement activity, a DNA strand
complimentary to the template DNAis synthesized, starting from the 3’-end of the F2 region

2. of the FIP. Outer primer F3 hybridize to the F3c region of the target DNA and extends,
displacing the forward inner primer (FIP) linked complimentary strand. This displaced strand
forms a loop at the 5’end.
3) This single stranded DNAwith a loop at the 5’end serves as the template for backward inner
primer (BIP).B2hybridizes to B2c region of the template DNA.DNAsynthesis is now initiated
leading to the formation of complimentary stand and opening of the 5’end loop.
4) Now, the outer primer B3 hybridizes to B3c region of the target DNAand extends, displacing
the BIP linked complimentary strand. This results in the formation of dumbbell shaped DNA.
5) The Nucleotides are added to the 3’end of F1 by DNApolymerase, which extends and opens
up the loop atthe 5’end. The dumbbell shaped DNAnow getsconverted to a stem loop structure.
This structure serves as an initiator for LAMP cycling, which is the second stage of the LAMP
reaction.
6) To initiate LAMP cycling, the FIP hybridizes to the loop of the stem-loop DNA structure. As
the FIP hybridizes to the loop, the F1 strand is displaced and form a new loop at 3’end.
7) Now nucleotides are added to the 3’end of B1.the extension takes place displacing the FIP
strand. This displaced strand again forms a dumbbell shaped DNA. Subsequent self-primed
strand displacement DNAsynthesis yield one complimentary structure of the original stem loop
DNAand one gap repaired stem loop DNA.
8) Bothe these products the serve as a template for a BIP primed strand displacement reaction in
the subsequent cycles.
5. Explain how CAR-T cell therapy works.
CAR T-cell therapy makes T cells focus their attention toward a substance the body thinks is harmful
called an antigen, which is found on the surface of specific cancer cells. In the production of CAR T
cells, a protein is added to the T cell’s surface to help them achieve this focus. This protein is called a
chimeric antigen receptor (CAR). When a T cell has a CAR added to it, it is called a “CAR T cell.”
CAR T cells work by floating around the body and looking for cells that carry the antigen programmed
into the CAR protein, like certain cancer cells. When a CAR T cell comes in contact with an antigen on
a cancercell, it activates.Activated CART cells multiply and signal to other parts of the immune system
to come to the site of the cancer cell. These signaling proteins are called cytokines. All of these
cytokines and activated T cells then causes significant inflammation focused at the cancer cell, which
causes the cancer cell to die. If all of the cancer cells die, the cancer can become in remission, which
means the cancer has disappeared either temporarily or permanently.
6. Explain how DNAdata storage works.
The digital data is encoded in a DNAsequence,the corresponding sequence information is synthesized
into an artificial DNAand the information is decoded by sequencing the artificial DNAstrand. This is
the exact path of storing and retrieving digital data from DNA. The DNAdata storage system involves
6 steps:(1) encoding: first, convert the digital code of 1’s and 0’s into the 4 building block of DNAcode
(A, C,T, and G,). 00 for A, 01 for G, 10 for C and 11 for T are the binary codes for storing information.
The information in the binary form is converted into long digital sequence of DNAcode of ATGC. (2)
synthesis and assembly: after the digital data is encoded in a DNA sequence, the corresponding
sequence information is synthesized into an artificial DNA. Uses various biological reactions to create
short sequencesof DNAand assemble them into longer strands.(3) storage: preserve the synthesis DNA
in a solution or (dried powder) form to minimize degradation overtime. (4) Retrieval: extracts subsets
of DNA from large sample. (5) sequencing: reads the series of DNA nucleotide base pairs through
techniques. (6) decoding: turns the base pair sequence into a binary stream (restoration of the original
digital information) by decoding and reassembling the data segments.
- Write the main point for each questions.
- Answer submission deadline: October 24, 2021.